Methods of indentifying modulators of the FGF receptor

ABSTRACT

The present invention provides fragments of SNT and FGFR which can form a binding complex that is amenable to structural determinations by NMR spectroscopy. The three-dimensional structural data is also included as part of the invention. In addition, the present invention provides methodology for related structure based rational drug design using the three-dimensional data. Nucleotide and amino acid sequences of the fragments are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a non-provisional application claiming thepriority of copending provisional U.S. Ser. No. 60/175,867 filed Jan.12, 2000, the disclosure of which is hereby incorporated by reference inits entirety. Applicants claim the benefits of this application under 35U.S.C. § 119(e).

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least inpart, by a grant from the National Institutes of Health, Grant No.GM59432-01. Accordingly, the Government may have certain rights in theinvention.

FIELD OF THE INVENTION

The present invention provides the three-dimensional structure of acomplex between the phosphotyrosine binding domain (PTB) of Suc1-associated neurotrophic factor target protein (SNT) and the SNTbinding site of the fibroblast growth factor receptor (FGFR). Thethree-dimensional structural information is included in the invention.The interaction between SNT and FGFR plays a key role in the regulationof cell proliferation. Therefore, the present invention providesprocedures for identifying agents that can inhibit tumor proliferationthrough the use of rational drug design predicated on thethree-dimensional data provided herein.

BACKGROUND OF THE INVENTION

The discovery and the extensive biochemical and biophysicalcharacterization of the evolutionarily conserved cytoplasmic proteinmodular domains (such as SH2, SH3, PH, and WW domains) haverevolutionized the way protein-protein interactions in cellular signaltransduction is understood [Pawson and Scott, Nature 278: 2075-2080(1997)]. Since these protein module-mediated interactions play animportant role in regulating numerous cellular processes including cellgrowth, proliferation, differentiation and apoptosis, studies of theprotein domain-mediated pathways have revealed the molecular basis ofvarious human diseases. This has led to the discovery of new drugtargets for treatment of these diseases.

The Phosphorylated Tyrosine Binding (PTB) domain (also called PID orSAIN domains) is another evolutionarily conserved cytoplasmic proteinmodular domain involved in cellular signal transduction. The PTB domainwas first identified as a protein module that could bind phosphorylatedtyrosines, and thus was classified as an alternative to the Src homology2 (SH2) domain [Blaikie et al. J. Biol. Chem. 269:32031-32034 (1994);Kavanaugh, and Williams, Science 266:862-1865 (1994); O'Neill et al.,Mol. Cell. Biol. 14: 6433-6442 (1994)]. However, PTB domains arestructurally and functionally distinct from SH2 domains, and recognizeamino acid residues amino-terminal (rather than carboxyl-terminal) tothe phosphotyrosine (pY) [Zhou and Fesik, S. W. Prog. Biophys. Molec.Biol. 64:221-235 (1995)]. In particular, PTB domains preferentially bindto phosphorylated proteins at sites containing an NPXpY motif andhydrophobic amino acids amino-terminal to this sequence [Gustafson etal. Mol. Cell. Biol. 15:2500-2508 (1995); Kavanaugh et al. Science268:1177-1179 (1995); Zhou et al., J. Biol. Chem. 270:31119-31123(1995); Trub et al., J. Biol. Chem. 270:18205-18208 (1995); Singyang, J.Biol. Chem. 270:14863-14866 (1995)]. Moreover, unlike SH2 domains, PTBdomains typically show very low protein sequence homology. Different PTBdomains exhibit distinct selectivity for residues amino-terminal to theNPXpY motif (SEQ ID NO:6). For example, the PTB domain of the insulinreceptor substrate 1. (IRS-1) favors hydrophobic residues at differentstructural locations of its receptor, than those preferred by the PTBdomain of the adaptor protein Shc. Recent studies demonstrate that thePTB domains of X11 and Numb can also recognize sequences related to theNPXpY motif in tyrosine-phosphorylation andnon-phosphorylation-dependent manners [Li et al. Proc. Natl. Acad. Sci.USA 94: 7204-7209 (1997); Li et al. Nat., Struct. Biol. 5:1075-1083(1998); and Zhang et al., EMBO J. 16:6141-6150 (1997)].

The NMR structural analyses of the Shc and IRS-1 PTB domains revealedthe detailed structural basis of their protein recognition [Zhou et al.,Nature 378:584-592 (1995); and Zhou et al, Nature Struct. Biol.3:388-393 (1996)]. Despite their very low sequence homology, the PTBdomains of Shc and IRS-1 consist of a conserved pleckstrin (PH) domainfold, i.e. a β-sandwich containing two nearly orthogonal, anti-parallelβ-sheets capped at one end by an amphipathic C-terminal α-helix.Furthermore as stated above, the structurally related PTB domains of Shcand IRS-1 employ two very different mechanisms for recognizing thephosphotyrosine and the hydrophobic residues amino-terminal to the NPXpYsequence. For example, for Shc, an Ile residue of a synthetic peptidederived from TRKA receptor (HIIENPQpYFSDA, SEQ ID NO:7) binds in a deephydrophobic pocket located between P5 and the C-terminal α-helix. Thecorresponding residue of a peptide derived from interleukin-4 receptor(IL-4R) (LVIAGNPApYRS, SEQ ID NO:4), binds on the surface of theprotein. In addition, the IRS-1 PTB domain recognizes Ile and Leu of theIL-4R peptide through interactions with a hydrophobic site on thesurface of a second β-sheet, whereas the analogous site in Shc is notavailable for peptide binding, because it is covered by a loop and theN-terminal portion of an α-helix. Notably, in contrast to SH2 domains,key arginines that are important for binding to phosphotyrosine arelocated in different regions of different PTB domain sequences. SNT(suc1-associated neurotrophic factor target protein) proteins are twonewly discovered insulin receptor substrate-like (IRS-like) signalingadaptor molecules that are specifically activated by receptors forfibroblast growth factors (FGF), nerve growth factors (NGF), andglial-derived neurotrophic factor, but are not activated by most othergrowth factor receptor kinases [Xu et al., J. Biol. Chem.273:17987-17990 (1998); Kouhara et al. Cell 89:693-702 (1997); Meakin etal., J. Biol. Chem. 274:9861-9870 (1999)]. SNT tyrosine phosphorylationpromotes activation of Ras/MAPK and SHP-2, two biochemical pathwayscritical for ligand-induced biological responses. Current studiessuggest that SNT activation enables FGFs and NGFs to elicit specificbiological responses not achieved by activation of other receptortyrosine kinases, which fail to stimulate SNTs. It has further beenshown that the activation and tyrosine-phosphorylation of SNTs requiredirect contact between receptors and the amino-terminal phosphotyrosinebinding domain of each SNT [Xu et al., J. Biol. Chem. 273:17987-17990(1998); Meakin et al., J. Biol. Chem. 274:9861-9870 (1999)]. Whereas SNTPTB domains recognize a canonical NPXpY motif on NGF receptors such asTRKA, they unexpectedly bind a tyrosine- and asparagine-free motif inthe juxtamembrane segment of the FGF receptor (FGFR). Thus, the SNT PTBdomain represents a very unique protein modular domain, which canspecifically bind two seemingly unrelated receptor peptide moieties.

The structural and functional diversity of PTB domains is furtherdemonstrated in the SNT PTB domains. Sequence homology alignment andsecondary prediction analysis using an approach of profile-based neuralnetwork predictions [PHD method; EMBL-Heidelberg et al., Mol. Bol. 232,584-599 (1993); Rost and Sander, Proteins 19:55-77 (1994)] reveals thatSNT PTB domains contain a large insert sequence (predicted to be anα-helix) located between the corresponding strand P7 and the C-terminalα-helix in the Shc and the IRS-1 PTB domains. It is interesting to notethat whereas in all of the published three-dimensional structures of PTBand PH domains (regardless of whether they were determined by NMR orX-ray crystallography) structural variations have been found indifferent loop regions, none of have been observed between the P7 andthe C-terminal α-helix region.

One means of modulating cellular proliferation and/or differentiation isto either inhibit or facilitate the interaction of the PTB domain of anSNT and the FGF receptor. Therefore, there is a need to identifyagonists or antagonists to the SNT/FGFR complex. Unfortunately, suchidentification has heretofore relied on serendipity and/or systematicscreening of large numbers of natural and synthetic compounds. A farsuperior method of drug-screening relies on structure based drug design.In this case, the three dimensional structure of SNT/FGFR complex isdetermined and potential agonists and/or potential antagonists aredesigned with the aid of computer modeling [Bugg et al., ScientificAmerican, December:92-98 (1993); West et al., TIPS, 16:67-74 (1995); andDunbrack et al., Folding & Design, 2:27-42 (1997)]. However, heretoforethe three-dimensional structure of the SNT/FGFR complex has remainedunknown. Therefore, there is a need for obtaining a form of the SNT/FGFRcomplex that is amenable for NMR analysis and/or X-ray crystallographicanalysis. Furthermore there is a need for the determination of thethree-dimensional structure of such complexes. Finally, there is a needfor procedures for related structural based drug design predicated onsuch structural data. The citation of any reference herein should not beconstrued as an admission that such reference is available as “PriorArt” to the instant application.

SUMMARY OF THE INVENTION

The present invention provides the structure of a binding complexbetween the PTB domain of SNT- 1 and a peptide derived from the SNT-1binding site of the fibroblast growth factor receptor (FGFR). Thestructural information provided can be employed in methods ofidentifying drugs that can modulate cellular proliferation since theSNT-1/FGFR interaction plays a major role in the regulation of mitogeniccell signaling. In a particular embodiment, the three-dimensionalstructural information is used in the design of an inhibitor of cellproliferation. Such an inhibitor could be used as an anti-tumor agent.

The present invention also provides an isolated nucleic acid thatencodes a polypeptide that comprises the amino acid residues 11-140 ofSEQ ID NO:1. In a related embodiment the peptide comprises amino acidresidues 1 1-140 of SEQ ID NO:1 with a conservative amino acidsubstitution.

The present invention also includes an isolated nucleic acid thatencodes a peptide derived from FGFR1 consisting of 12 to 100 aminoacids, preferably 16 to 50 amino acids and more preferably 20 to 30amino acids which comprises the amino acid sequence of SEQ ID NO:5:

-   -   Val Xaa Xaa Leu Xaa Xaa Xaa Ile Xaa Leu Xaa Arg Xaa Val Xaa Val.        Preferably this peptide binds to the PTB domain of SNT1.

In another embodiment, the present invention provides an isolatednucleic acid that encodes a peptide derived from FGFR1 consisting of 12to 100 amino acids, preferably 16 to 50 amino acids and more preferably20 to 30 amino acids which comprises the amino acid sequence of SEQ IDNO:3. In a related embodiment the nucleic acid encodes a peptide thatcomprises the amino acid sequence of SEQ ID NO:3 with a conservativeamino acid substitution. Again it is preferable that the peptide bindsto the PTB domain of SNT1.

All of the nucleic acids of the present invention can be in an isolatedform, and/or operatively linked to an expression control sequence. Inaddition, all of the nucleic acids of the present invention can furthercomprise a heterologous nucleotide sequence. Furthermore, all of thenucleic acids of the present invention operatively linked to anexpression control sequence can be used to transform or transfect aunicellular host. The present invention also provides methods ofexpressing the peptides and proteins fragments of the present inventionin the unicellular host. One such method comprises culturing theunicellular host in an appropriate cell culture medium under conditionsthat provide for the expression of the fragment and/or peptide by thecell. In addition, the present invention includes methods that furthercomprise the step of purifying the peptides and fragments. The purifiedforms of the fragments and peptides are also included as part of thepresent invention.

The present invention also provides an isolated polypeptide comprisingamino acid residues 11-140 of SEQ ID NO:1. In a related embodiment thepeptide comprises amino acid residues 11-140 of SEQ ID NO:1 with aconservative amino acid substitution.

The present invention also includes an isolated peptide derived fromFGFR1 consisting of 12 to 100 amino acids, preferably 16 to 50 aminoacids and more preferably 20 to 30 amino acids which comprises the aminoacid sequence of SEQ ID NO:5:

-   -   Val Xaa Xaa Leu Xaa Xaa Xaa Ile Xaa Leu Xaa Arg Xaa Val Xaa Val.        Preferably this peptide binds to the PTB domain of SNT1.

In another embodiment, the present invention includes an isolatedpeptide derived from FGFR1 consisting of 12 to 100 amino acids,preferably 16 to 50 amino acids and more preferably 20 to 30 amino acidswhich comprises the amino acid sequence of SEQ ID NO:3. In a relatedembodiment the peptide comprises the amino acid sequence of SEQ ID NO:3with a conservative amino acid substitution. Again it is preferable thatthe peptide binds to the PTB domain of SNT1. In a particular embodiment,the peptide has the amino acid sequence of SEQ ID NO:4.

The present invention also provides fusion proteins/peptides includingchimeric proteins/peptides comprising the peptides and/or fragments ofthe present invention. All of the isolated peptides and/or fragments ofthe present invention, and all of the recombinant peptides and/orfragments of the present invention may be parts of these fusion andchimeric proteins/peptides. In one such embodiment, a fusion proteincomprises the PTB domain of SNT-1 and the green fluorescent protein. Inanother such embodiment, the fusion protein comprises a FGFR derivedpeptide together with a FLAG tag. The present invention further providesmethods of using the peptides and/or fragments of the present inventionin a drug screening assay. Any of the peptides and/or fragments, and/orfusion proteins/peptides of the present invention may be used in suchmethods. The peptides or protein fragments of the present invention canalso be labeled. In another such embodiment, the peptides and/orfragments can be bound to a solid support.

The present invention further provides assays for testing potentialdrugs that are selected/identified by the three-dimensional structuralanalysis of SNT/FGFR complex of the present invention, for the abilityof the potential drug to interfere with the complex formation, forexample.

The present invention further provides methods of identifying compoundsor agents that modulate the stability of a SNT/FGFR complex and/ormodulate the binding of SNT with FGFR. In one aspect of the presentinvention these methods employ the three-dimensional structure of theSNT/FGFR complex.

One such embodiment comprises selecting a potential compound byperforming rational drug design with the set of atomic coordinatesobtained from Tables 1-5. In a related embodiment the selection of thepotential compound is performed by rational drug design after obtaininga set of atomic coordinates defining the three-dimensional structure ofthe SNT/FGFR complex consisting of a fragment of SNT consisting of aminoacid residues 11-140 of SEQ ID NO:1 and a fragment of FGFR consisting ofSEQ ID NO:3. Preferably the selecting is performed in conjunction withcomputer modeling. In either case, the potential compound is contactedwith a SNT/FGFR complex comprising an SNT or an SNT fragment, and FGFRor an FGFR fragment, and the stability of the SNT/FGFR complex isdetermined (e.g., measured) in the presence of the compound. A potentialcompound is identified as a compound that modulates the stability of theSNT/FGFR complex when there is an change in the stability of theSNT/FGFR complex. The compound is identified as a stabilizer of theSNT/FGFR complex when the stability of the SNT/FGFR complex increases inits presence, whereas the compound is identified as a destabilizer ofthe SNT/FGFR complex when the stability of the SNT/FGFR complexdecreases in its presence.

In another embodiment, the method identifies a compound or an agent thatmodulates the formation of a SNT/FGFR complex using thethree-dimensional structure of the SNT/FGFR complex. One particularembodiment of this type comprises selecting a potential compound thatbinds to the PTB domain of SNT. In another embodiment, a potentialcompound is selected that binds to the SNT binding region of FGFR.Preferably the selection is performed using rational drug design withthe set of atomic coordinates obtained from Tables 1-5. Alternatively,the selection of the potential compound is performed by rational drugdesign after obtaining a set of atomic coordinates defining thethree-dimensional structure of the SNT/FGFR complex consisting of afragment of SNT consisting of amino acid residues 11-140 of SEQ ID NO:1and a fragment of FGFR consisting of SEQ ID NO:3. Preferably theselection is performed in conjunction with computer modeling. Thepotential compound is contacted with an SNT or an SNT fragment, and FGFRor an FGFR fragment under conditions in which the SNT/FGFR complex canform in the absence of the potential compound. The binding affinity ofthe SNT or the SNT fragment with FGFR or the FGFR fragment is thendetermined (e.g., measured) in the presence of the compound. A potentialcompound is identified as a compound that modulates the formation of theSNT/FGFR complex when there is a change in the binding affinity of theSNT or the SNT fragment with FGFR or the FGFR fragment. The compound isidentified as an agonist of the formation of the SNT/FGFR complex whenthe binding affinity of the SNT/FGFR complex increases in its presence,whereas the compound is identified as an inhibitor of the formation ofthe SNT/FGFR complex when the binding affinity of the SNT/FGFR complexdecreases in its presence.

The present invention further provides a method of selecting a compoundor an agent that potentially modulates the SNT/FGFR dependent cellularsignaling pathway. One such embodiment comprises defining the structureof the SNT/FGFR complex by the atomic coordinates obtained from Tables1-5 and selecting a compound which potentially modulates the SNT/FGFRdependent cellular signaling pathway with the aid of the definedstructure. In a related embodiment the selection of the potentialcompound is performed by rational drug design after obtaining a set ofatomic coordinates defining the three-dimensional structure of theSNT/FGFR complex consisting of a fragment of SNT consisting of aminoacid residues 11-140 of SEQ ID NO:1 and a fragment of FGFR consisting ofSEQ ID NO:3. In one such embodiment the compound inhibits the SNT/FGFRdependent cellular signaling pathway, whereas in another embodiment thecompound stimulates the SNT/FGFR dependent cellular signaling pathway.

The present invention further provides a method of selecting a compoundthat potentially binds to the PTB domain of SNT1 that comprises definingthe structure of the SNT/FGFR complex by the atomic coordinates obtainedfrom Tables 1-5. With the aid of the defined structure a compound isselected which potentially binds the PTB domain of SNT1. In a relatedembodiment the selection of the potential compound is performed byrational drug design after obtaining a set of atomic coordinatesdefining the three-dimensional structure of the SNT/FGFR complexconsisting of a fragment of SNT consisting of amino acid residues 1-140of SEQ ID NO:1 and a fragment of FGFR consisting of SEQ ID NO:3. In arelated embodiment, a compound is selected that binds to the SNT bindingregion of FGFR. In another embodiment, the compound is selected to bindto the SNT/FGFR complex.

As anyone having skill in the art of drug development would readilyunderstand, the potential drugs selected by the above methodologies canbe refined by re-testing in appropriate drug assays, including thosedisclosed herein. Chemical analogs of such potential drugs can beobtained (either through chemical synthesis or drug libraries) and beanalogously tested. Therefore, methods comprising successive iterationsof the steps of the individual drug assays, as exemplified herein, usingeither repetitive or different binding studies, or transcriptionactivation studies or other such studies are envisioned in the presentinvention. In addition, potential drugs may be identified first by rapidthroughput drug screening, as described below, prior to performingcomputer modeling on a potential drug using the three-dimensionalstructure of the SNT/FGFR complex.

The present invention further comprises all of the potential, selected,and putative drugs as well as the drugs themselves identified by methodsof the present invention. Accordingly, it is a principal object of thepresent invention to provide the three-dimensional coordinates of theSNT/FGFR complex.

It is a further object of the present invention to provide solublefragments of SNT that comprise the PTB domain, and that can bind toFGFR.

It is a further object of the present invention to provide a solublefragment of FGFR that comprises The binding site for the PTB of SNT.

It is a further object of the present invention to provide methods ofidentifying drugs that can modulate the SNT/FGFR interaction.

It is a further object of the present invention to provide methods ofidentifying drugs that can modulate cellular proliferation throughmodulating the SNT/FGFR interaction.

It is a further object of the present invention to provide methods thatincorporate the use of rational design for identifying drugs thatstabilize the SNT/FGFR complex.

It is a further object of the present invention to provide methods thatincorporate the use of rational design for identifying drugs thatinhibit the SNT/FGFR complex.

It is a further object of the present invention to provide detailedstructural information regarding the binding of SNT and FGFRelectronically, magnetically, or electromagnetically.

It is a further object of the present invention to provide a computerthat comprises a representation of the three-dimensional structure ofthe SNT/FGFR binding complex and/or relevant portions thereof.

It is a further object of the present invention to provide methodologiesfor exploiting such structural information in order to develop potentialanti-tumor drugs with the use of structure based rational drug design.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict an alignment of protein sequence homology. FIG. 1Adepicts the Sequence alignment of PTB domains of the SNT and IRSproteins. Amino acid sequence and accession numbers of the proteins areindicated along the protein sequences. Protein sequences of FRS2α andFRS2β have been reported [Ong et al., Mol. Cell. Biol. 20:979-989(2000)]. The experimentally determined secondary structure elements aredisplayed above or below the sequences of the PTB domains of SNTs orIRSs [Zhou et al., Nat. Struc. Biol. 3:388-393 (1996)], respectively.Asterisks highlight residues in the SNT-1 PTB domain that showintermolecular NOEs to the hFGFR1 peptide. Absolutely or highlyconserved residues among the SNT and IRS PTB domains are shown in redand blue, respectively. Two underlined Arg residues of SNT-1 were bothchanged by site-directed mutagenesis to Gln. Arrows indicate constructsused in truncation analysis of SNT-1 PTB domain binding to hFGFR1 orTRK. Pro residues located C-terminal to the SNT-1 PTB domain are shownin bold. FIG. 1B depicts the Sequence alignment of the juxtamembraneregion of the FGFR family. For each FGFR group (FGFR1-4), proteinsequences from three representative species, i.e., human, mouse, andxenopus, are selected. The number of observed intermolecular NOEsidentified for a particular amino acid residue of the hFGFR1 peptide isshown in red above the sequence. Absolutely or highly conserved residuesare highlighted in yellow and blue background, respectively.

FIGS. 2A-2D depict the structure of the SNT-1 PTB domain/hFGFR1 complex.FIG. 2A shows the stereoview of the backbone atom superposition of thefinal 20 NMR-derived structures of the complex, containing the SNT-1 PTBdomain residues 18-116 and the hFGFR1 peptide residues 411-430. Theterminal residues, which are structurally disordered, are omitted forclarity. For the final 20 structures, the root-mean-square deviations(RMSDs) of the backbone and all heavy atoms for protein residues 18-116are 0.74±0.16 Å and 1.46±0.16 Å, respectively. The corresponding RMSDsfor the protein secondary structure regions (protein residues 19-24,35-40, 45-49, 52-57, 63-68, 71-76, 85-90, 94-107 and 111-115) are0.40±0.05 Å and 0.88±0.05 Å, respectively. The RMSDs of the backbone andall heavy atoms for the hFGFR1 peptide (residues 412-430) are 0.56±0.10Å and 1.25±0.15 Å, respectively. FIG. 2B is a ribbon depiction of theaveraged minimized NMR structure of the SNT-1 PTB domain/hFGFk1 complex.The orientation of FIG. 2B is as shown in FIG. 2A. FIG. 2C is a ribbondiagram of the SNT-1 PTB domain structure from the top of the protein,which is rotated ˜90° from the orientation in FIG. 2B. FIG. 2D is amolecular surface representation of the SNT-1 PTB domain structurecalculated in GRASP [Nicholls et al., Biophys. J. 64:166-170 (1993)].The protein is color-coded by surface curvature, and the color gradientfrom green to dark gray reflects decreasing solvent exposure. The hFGFR1peptide molecule is shown as a ball and stick representation color-codedby atom-type.

FIG. 3A depicts the 2D ¹H/¹⁵N-HSQC spectrum of the SNT-1 PTB domain(0.25 mM) complexed to a FGFR peptide (1:1). The spectrum was recordedon a Bruker DRX 500 MHz NMR spectrometer for the protein in 50 mMphosphate buffer of pH 6.5 containing 5 mM DTT and 0.5 mM EDTA at 25° C.FIG. 3B depicts the effects of a FGFR peptide or a TRKA phosphopeptidebinding on the backbone amide signals in the HSQC spectrum of the SNT-1PTB domain. The amide peaks of the protein in the free, the FGFRpeptide-, or the TRKA phosphopeptide-complexed forms are color-coded inblack, red, and blue, respectively. Green arrows indicate the shifts ofthe amide signals upon binding to the corresponding peptides.

FIGS. 4A-4E depict the intermolecular interactions in the SNT-1 PTBdomain/hFGFR1 complex. FIG. 4 depicts the secondary structure of theintermolecular antiparallel β-sheet of the complex. The number ofintermolecular NOEs observed in ¹³C- or ¹⁵N-edited (F₁),¹³C/¹⁵N-filtered (F₃) 3D NOESY spectra is summarized for individualamino acid residue. NOEs that define the structure of the β-sheet areindicated by arrows. Arrows for intramolecular, ¹³C-based and 15N-basedintermolecular NOEs, are color-coded in black, green and red,respectively. Broken lines (blue) highlight two intermolecular hydrogenbonds that are supported by amide exchange data. The intermolecularinteractions are depicted for three-regions of the hFGFR1 peptide(green). FIG. 4B depicts the C-terminal (residues 424-430) region. Theside-chains of the protein and the peptide residues are displayed inorange and blue, respectively. FIG. 4C shows the middle (residues417-423) region. The three loops connecting β1/β2, β3/β4 and β6/β7 thatform a hydrophobic binding pocket for binding to the peptide residueVal-414 are colored in pink. FIG. 4D shows the N-terminal (residues409-416) region. FIG. 4E depicts the complementary electrostaticinteractions between the SNT-1 PTB domain and hFGFR1 peptide residues.

FIG. 5 depicts a schematic of a computer comprising a central processingunit (“CPU”), a working memory, a mass storage memory, a displayterminal, and a keyboard that are interconnected by a conventionalbidirectional system bus. The computer can be used to display andmanipulate the structural data of the present invention.

FIGS. 6A-6D show the mutational analysis of the SNT-1 PTB domaininteractions with FGFRs or TRKs. FIG. 6A shows the effect of hFGFR1point mutations on interactions with the SNT-1 PTB domain, determined byyeast two-hybrid binding assays. Data for peptide mutants are calculatedfrom an average of 5 independent experiments. Western blot showing BDfusion protein expression of wild type and mutant hFGFR1 in the yeastcells. FIG. 6B shows the structure of the SNT-1 PTB domain/hFGFR1complex showing location of Arg-63 and Arg-78 (blue) that are essentialfor binding to the phosphotyrosine in the NPXpY motif. The backbone ofthe hFGFR1 peptide is shown in green. The distinct β8 strand of theSNT-1 PTB domain is displayed in red. FIG. 6C depicts the structure ofthe IRS-1 PTB domain in complex with a tyrosine-phosphorylated peptidederived from interleukin-4 receptor (LVIAGNPApYRS, residues 489-499)determined by NMR [Zhou et al., Nat. Struc, Biol. 3:388-393 (1996)]. Thepeptide residues are shown in green, and the two key Arg residues(Arg-212 and Arg-227) of the PTB domain that are essential forphosphotyrosine binding are displayed in blue. FIG. 6D shows the resultsof a yeast two-hybrid binding studies of SNT-1 β8 truncation effect onits interactions with hFGFR1 and tyrosine-phosphorylated TRKB. The panelframed in red shows the loss of interaction between hFGFR1 and the SNT-1PTB domain protein lacking the β8 strand. Colony formation on thesynthetic complete medium lacking Leu and Trp (Leu⁻, Trp⁻) illustratesthe efficiency of co-transformation with the two plasmids, while growthon the corresponding medium lacking His, Leu and Trp (His⁻, Leu⁻, Trp⁻)shows level of protein-protein interaction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the first detailed structural informationregarding the complex of the PTB domain of SNT-1 with a peptide derivedfrom the known SNT-1 binding site of the fibroblast growth factorreceptor (FGFR). The present invention therefore provides thethree-dimensional structure of the SNT-1 PTB domain/FGFR complex. Sincethe SNT-1/FGFR interaction plays a significant role in the regulation ofmitogenic cell signaling, the structural information provided herein canbe employed in methods of identifying drugs that can modulate theproliferation of cells. In a particular embodiment, thethree-dimensional structural information is used in the design of aninhibitor of cell proliferation for the treatment of cancer.

The three-dimensional structure of the SNT-1 PTB domain/FGFR complexdisclosed herein demonstrates that the interaction between the PTBdomain of SNT-1 and FGFR differs significantly from the manner that PTBdomains have been reported to interact with the canonical NPXpY motif(where X is any amino acid and pY is a phosphorylated tyrosine).Furthermore, the structural information disclosed herein provides thebasis for mutational analysis of the SNT-1 PTB/FGFR complex. Key aminoacid residues from both SNT-1 and FGFR that are essential for theprotein-protein recognition are also identified.

SNT-1 also plays a role in regulating nerve growth factor (NGF) receptorsignaling via its binding through its PTB domain with theactivated/tyrosine phosphorylated NGF receptor. As stated above thisinteraction differs significantly with that disclosed herein for theSNT/FGFR complex. Based on the new structural information, the key aminoacid residues for the binding of the SNT1-PTB domain with the NGF TRKAreceptor can be identified and further elucidated using basicmutagenesis and standard isothermal titration calorimetry. The resultsobtained from the structural and functional studies disclosed hereinindicate that there is a novel mechanism through which SNT proteinsregulate neuronal cell growth and differentiation. Such detailedstructural and mechanistic findings provide the foundation forstructure-based rational drug design. The agents identified by thisprocedure will be useful for ameliorating conditions involving thedysfunction of cell signaling pathways involving the NGF receptor andthe FGF receptor. More particularly, FGFRs have been implicated to playa causal role in the development of the following human ailments such asin tumorigenesis including various forms of human cancer, and inskeletal disorders, such as Achondroplasia, hypochondroplasia, Crouzonsyndrome, Apert syndrome, and Pfeiffer syndrome.

Structure based rational drug design is the most efficient method ofdrug development. However, heretofore, little information has beendisclosed regarding the structure of the SNT/FGFR interaction. Obtainingdetailed structural information requires an extensive NMR or X-raycrystallographic analysis. In the former case, the entire SNT-1/FGFRcomplex has a molecular weight which is beyond the present capabilitiesof NMR analysis. In the latter case, crystallography of proteins remainsa black art. The present invention overcomes the difficulties describedabove, by providing fragments of the SNT-1/FGFR complex (identifiedbelow) that retain the necessary structural elements for binding. SuchSNT-1/FGFR complexes are amenable to NMR structural analysis. Bydetermining and then exploiting the detailed structural information ofthis complex (exemplified by NMR analysis below) the present inventionprovides novel methods for developing new anti-tumor drugs throughstructure based rational drug design.

Thus the present invention provides a representative set of structuralcoordinates for the SNT-1 PTB domain/FGFR peptide complex (Table 1)which were obtained by NMR analysis. A Ribbon diagrams of thethree-dimensional structure of the SNT-1 PTB domain in complex with theFGFR peptide is shown in FIGS. 2 and 4. The present invention alsoprovides the NOE-derived distance restraints, and NMR chemical shiftassignments of the SNT-1 PTB domain/FGFR peptide. The NMR chemical shiftassignments of the SNT-1 PTB domain/FGFR peptide complex are included inthe chemical shift table (Table 2) for the ¹H-¹⁵N HSQC spectrum of SNT-1PTB domain/FGFR peptide complex. Tables 3-5 contain the NMRexperimentally determined distance restraints that are used in thestructural calculations, including the hydrogen bond distance restraints(Table 3), the unambiguous distance restraints

(Table 4) and the ambiguous distance restraints (Table 5). The samplecoordinates data set of Table 1, the chemical shifts of Table 2, alongwith the information contained in Tables 3-5 are sufficient to enablethe skilled artisan to practice the invention. In addition, Tables 1-5are also capable of being placed into a computer readable form which isalso part of the present invention. Furthermore, methods of using thesecoordinates and chemical shifts and related information (including incomputer readable forms) in drug assays are disclosed. Moreparticularly, such coordinates can be used to identify potential ligandsor drugs which will modulate the binding of the SNT with FGFR. Since theSNT/FGFR interaction is naturally stimulated by FGF and leads to theactivation of cell proliferation agents such as Ras, compounds thatinhibit the SNT FGFR binding will act to inhibit cellular proliferationat a comparatively early stage of the signal transduction pathway.Similarly, compounds that act as agonists for the SNT FGFR interactionwill act as agonists for cellular proliferation.

In addition, the present invention provides a computer that comprises arepresentation of the SNT/FGFR binding complex in computer memory thatcan be used to screen for compounds that will enhance or alternativelyinhibit the binding of SNT to FGFR. In a related embodiment, thecomputer can be used in the design of altered SNT and/or FGFR proteinsthat have either enhanced, or alternatively diminished binding affinityfor each other. Preferably, the computer comprises portions or all ofthe information contained in Tables 1-5. In a particular embodiment, thecomputer comprises: (i) a machine-readable data storage material encodedwith machine-readable data, (ii) a working memory for storinginstructions for processing the machine readable data, (iii) a centralprocessing unit coupled to the working memory and the machine-readabledata storage material for processing the machine-readable data into athree-dimensional representation, and (iv) a display coupled to thecentral processing unit for displaying the three-dimensionalrepresentation.

Thus the machine-readable data storage medium comprises a data storagematerial encoded with machine readable data which can comprise portionsor all of the structural information contained in Tables 1-5. Oneembodiment for manipulating and displaying the structural data providedby the present invention is schematically depicted in FIG. 5. Asdepicted the System 1, includes a computer 2 comprising a centralprocessing unit (“CPU”) 3, a working memory 4 which may be random-accessmemory or “core” memory, mass storage memory 5 (e.g., one or more diskor CD-ROM drives), a display terminal 6 (e.g., a cathode-ray tube), oneor more keyboards 7, one or more input lines 10, and one or more outputlines 20, all of which are interconnected by a conventionalbidirectional system bus 30.

Input hardware 12, coupled to the computer 2 by input lines 10, may beimplemented in a variety of ways. Machine-readable data may be inputtedvia the use of one or more modems 14 connected by a telephone line ordedicated data line 16. Alternatively or additionally, the inputhardware 12 may comprise CD-ROM or disk drives 5. In conjunction withthe display terminal 6, the keyboard 7 may also be used as an inputdevice. Output hardware 22, coupled to computer 2 by output lines 20,may similarly be implemented by conventional devices. Output hardware 22may include a display terminal 6 for displaying the three dimensionaldata. Output hardware might also include a printer 24, so that a hardcopy output may be produced, or a disk drive 5, to store system outputfor later use, see also U.S. Pat. No.: 5,978,740, Issued Nov. 2, 1999,the contents of which are hereby incorporated by reference in theirentireties.

In operation, the CPU 3 (i) coordinates the use of the various input andoutput devices 12 and 22; (ii) coordinates data accesses from massstorage 5 and accesses to and from working memory 4; and (iii)determines the sequence of data processing steps. Any of a number ofprograms may be used to process the machine-readable data of thisinvention.

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

As used herein the term “SNT-1 PTB domain/FGFR peptide complex” is usedinterchangeably with the term “SNT/FGFR complex” and the term “SNT-FGFRcomplex” are all meant to denote a binding complex between the PTBdomain of SNT or portion thereof that binds to the FGF receptor, and afragment of the FGF receptor that binds to the PTB domain of SNT. Onesuch a complex is identified in the Example below.

As used herein the term “SNT/FGFR dependent cellular signaling pathway”is a cellular signaling pathway in which the direct interaction betweenSNT and the FGF receptor (i.e., binding) is involved in transmitting thesignal from an extracellular ligand for the receptor to the nucleus ofthe cell.

A “polypeptide” comprising a fragment of FGFR or SNT (or moreparticularly the SNT PTB domain) as used herein can be the “fragment”alone, or a larger chimeric or fusion peptide/protein which contains the“fragment”.

As used herein a polypeptide or peptide “consisting essentially of” orthat “consists essentially of” a specified amino acid sequence is apolypeptide or peptide that retains the general characteristics, e.g.,activity of the polypeptide or peptide having the specified amino acidsequence and is otherwise identical to that protein in amino acidsequence except it consists of plus or minus 10% or fewer, preferablyplus or minus 5% or fewer, and more preferably plus or minus 2.5% orfewer amino acid residues.

As used herein the terms “fusion protein” and “fusion peptide” are usedinterchangeably and encompass “chimeric proteins and/or chimericpeptides” and fusion “intein proteins/peptides”. A fusion protein of thepresent invention can comprises a portion of an SNT or FGFR of thepresent invention joined via a peptide bond to at least a portion ofanother protein or peptide including a second SNT or FGFR protein in achimeric fusion protein. For example fusion proteins can comprise amarker protein or peptide, or a protein or peptide that aids in theidentification and/or monitoring of an SNT or FGFR peptide/protein ofthe present invention.

As used herein the terms “approximately” and “about” are used to signifythat a value is within ten percent of the indicated value i.e., aprotein fragment containing “approximately” 140 amino acid residues cancontain between 126 and 154 amino acid residues.

As used herein the term “binds to” is meant to include all such specificinteractions that result in two or more molecules showing a preferencefor one another relative to some third molecule. This includes processessuch as covalent, ionic, hydrophobic and hydrogen bonding but does notinclude non-specific associations such solvent preferences.

As used herein, and unless otherwise specified, the terms “agent”,“potential drug”, “test compound” or “potential compound” are usedinterchangeably, and refer to chemicals which potentially have a use asan inhibitor or activator/stabilizer of SNT/FGFR binding, and preferablyinclude drugs for the treatment or prevention of a disease and/orcondition involving the FGF receptor. Therefore, such “agents”,“potential drugs”, and “potential compounds” may be used, as describedherein, in drug assays and drug screens and the like.

As used herein a “small organic molecule” is an organic compound [ororganic compound complexed with an inorganic compound (e.g., metal)]that has a molecular weight of less than 3 Kilodaltons.

As used herein, the term “gene” refers to an assembly of nucleotidesthat encode a polypeptide, and includes cDNA and genomic DNA nucleicacids.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “replicon” is any genetic element (e.g.,plasmid, chromosome, virus) that functions as an autonomous unit of DNAreplication in vivo, i.e., capable of replication under its own control.

A “cassette” refers to a segment of DNA that can be inserted into avector at specific restriction sites. The segment of DNA encodes apolypeptide of interest, and the cassette and restriction sites aredesigned to ensure insertion of the cassette in the proper reading framefor transcription and translation.

A cell has been “transfected” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell.

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogues thereof such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk; and no formamide;or 30%, formamide, 5×SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6×SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6×SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-10.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-1 1.8). Preferably a minimum length for ahybridizable nucleic acid is at least about 12 nucleotides; preferablyat least about 18 nucleotides; and more preferably the length is atleast about 27 nucleotides; and most preferably 36 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a T_(m) of 55° C., and utilizes conditions as set forth above.In a preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C. Preferably the wash and hybridization(binding) steps are identical.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in a cell in vitro or invivo when placed under the control of appropriate regulatory sequences.The boundaries of the coding sequence are determined by a start codon atthe 5′ (amino) terminus and a translation stop codon at the 3′(carboxyl) terminus. A coding sequence can include, but is not limitedto, prokaryotic sequences and synthetic DNA sequences. If the codingsequence is intended for expression in a eukaryotic cell, apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence. Transcriptional andtranslational control sequences are DNA regulatory sequences, such aspromoters, enhancers, terminators, and the like, that provide for theexpression of a coding sequence in a host cell. In eukaryotic cells,polyadenylation signals are control sequences.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

As used herein, the term “homologous” in all its grammatical formsrefers to the relationship between proteins that possess a “commonevolutionary origin,” including proteins from superfamilies (e.g., theimmunoglobulin superfamily) and homologous proteins from differentspecies (e.g., myosin light chain, etc.) [Reeck et al., Cell, 50:667(1987)]. Such proteins have sequence homology as reflected by their highdegree of sequence similarity.

In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 50%(preferably at least about 75%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

Similarly, in a particular embodiment, two amino acid sequences are“substantially homologous” or “substantially similar” when greater than25% of the amino acids are identical (preferably at least about 50%,more preferably at least about 75%, and most preferably at least about90 or 95% identical), or greater than about 60% (preferably at leastabout 75%, more preferably at least about 90%, and most preferably atleast about 95 or 100%) are functionally identical. The sequencecomparison is performed over a contiguous block of amino acid residuescomprised by SNT, for example. In a preferred embodiment selecteddeletions or insertions that could otherwise alter the correspondencebetween the two amino acid sequences are taken into account. Preferablystandard computer analysis is employed for the determination that iscomparable, (or identical) to that determined with an Advanced Blastsearch at www.ncbi.nlm.nih.gov under the default filter conditions[e.g., using the GCG (Genetics Computer Group, Program Manual for theGCG Package, Version 7, Madison, Wisc.) pileup program using the defaultparameters].

As used herein an amino acid sequence is 100% “homologous” to a secondamino acid sequence if the two amino acid sequences are identical,and/or differ only by neutral or conservative substitutions as definedbelow. Accordingly, an amino acid sequence is 50% “homologous” to asecond amino acid sequence if 50% of the two amino acid sequences areidentical, and/or differ only by neutral or conservative substitutions.

The term “corresponding to” is used herein to refer similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

As used herein a “heterologous nucleotide sequence” is a nucleotidesequence that is added to a nucleotide sequence of the present inventionby recombinant methods to form a nucleic acid which is not naturallyformed in nature. Such nucleic acids can encode fusion proteins orpeptides, including chimeric proteins and peptides. Thus theheterologous nucleotide sequence can encode peptides and/or proteinswhich contain regulatory and/or structural properties. In another suchembodiment the heterologous nucleotide can encode a protein or peptidethat functions as a means of detecting the protein or peptide encoded bythe nucleotide sequence of the present invention after the recombinantnucleic acid is expressed. In still another such embodiment theheterologous nucleotide can function as a means of detecting anucleotide sequence of the present invention. A heterologous nucleotidesequence can comprise non-coding sequences including restriction sites,regulatory sites, promoters and the like.

General Techniques for Constructing Nucleic Acids that Express the SNTor FGFR Fragments of the Present Invention.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

The present invention also relates to cloning vectors containing nucleicacids encoding analogs and derivatives of the SNT and FGFR fragments ofthe present invention, including modified fragments, that have the sameor homologous functional activity as the individual fragments, andhomologs thereof The production and use of derivatives and analogsrelated to the fragments are within the scope of the present invention.

Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence as anucleic acid encoding an FGFR fragment or peptide, or SNT PTB domain ofthe present invention may be used in the practice of the presentinvention. These include but are not limited to allelic genes,homologous genes from other species, which are altered by thesubstitution of different codons that encode the same amino acid residuewithin the sequence, thus producing a silent change. Likewise the FGFRfragments or peptides, or SNT PTB domains of the invention include, butare not limited to, those containing, as a primary amino acid sequence,analogous portions of their respective amino acid sequences includingaltered sequences in which functionally equivalent amino acid residuesare substituted for residues within the sequence resulting in aconservative amino acid substitution. For example, one or more aminoacid residues within the sequence can be substituted by another aminoacid of a similar polarity, which acts as a functional equivalent,resulting in a silent alteration. Substitutes for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs. For example, the nonpolar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. Amino acids containingaromatic ring structures are phenylalanine, tryptophan, and tyrosine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, and lysine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid.

Particularly preferred conserved amino acid exchanges are:

(a) Lys for Arg or vice versa such that a positive charge may bemaintained;

(b) Glu for Asp or vice versa such that a negative charge may bemaintained;

(c) Ser for Thr or vice versa such that a free —OH can be maintained;

(d) Gln for Asn or vice versa such that a free NH₂ can be maintained;

(e) Ile for Leu or for Val or vice versa as roughly equivalenthydrophobic amino acids; and

(f) Phe for Tyr or vice versa as roughly equivalent aromatic aminoacids.

A conservative change generally leads to less change in the structureand function of the resulting protein. A non-conservative change is morelikely to alter the structure, activity or function of the resultingprotein. The present invention should be considered to include sequencescontaining conservative changes which do not significantly alter theactivity or binding characteristics of the resulting protein. Specificamino acid residues for both the SNT and the FGFR fragments have beenidentified that are important for binding, indicating a lower stringencyfor the substitution of the remaining amino acids residues.

All of the FGFR and SNT PTB domain peptides/fragments of the presentinvention can be modified by being placed in a fusion or chimericpeptide or protein, or labeled e.g., to have an N-terminal FLAG-tag, orHis6 tag. In a particular embodiment the SNT PTB domain fragment can bemodified to contain a marker protein such as green fluorescent proteinas described in U.S. Pat. No. 5,625,048 filed Apr. 29, 1997 and WO97/26333, published Jul. 24, 1997 each of which are hereby incorporatedby reference herein in their entireties.

The nucleic acids encoding peptides and protein fragments of the presentinvention and analogs thereof can be produced by various methods knownin the art. The manipulations which result in their production can occurat the gene or protein level [Sambrook et al., 1989, supra]. Thenucleotide sequence can be cleaved at appropriate sites with restrictionendonuclease(s), followed by further enzymatic modification if desired,isolated, and ligated in vitro. In addition a nucleic acid sequence canbe mutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypreexisting ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, in vitro site-directed mutagenesis [Hutchinson et al.,J. Biol. Chem., 253:6551 (1978); Zoller and Smith, DNA, 3:479-488(1984); Oliphant et al., Gene, 44:177 (1986); Hutchinson et al., Proc.Natl. Acad. Sci. U.S.A., 83:710 (1986)], use of TAB® linkers(Pharmacia), etc. PCR techniques are preferred for site directedmutagenesis [see Higuchi, 1989, “Using PCR to Engineer DNA”, in PCRTechnology: Principles and Applications for DNA Amplification, H.Erlich, ed., Stockton Press, Chapter 6, pp. 61-70].

The identified and isolated nucleic acids can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used.

Protein Expression and Purification

A bacterial protein expression system can be used to make various stableisotopically labeled (¹³C, ¹⁵N, and ²H) protein samples that are usefulfor a three-dimensional NMR structural determination of a proteincomplex. For example a pET15b (Novagen) bacterial expression vector canbe constructed which expresses the recombinant SNT-1 PTB domain as anamino-terminal His-tagged fusion protein.

Protein expression and purification can be conducted using standardprocedures for His-tagged proteins [Zhou et al., J. Biol. Chem.270:31119-31123 (1995)]. To optimize the level of protein expression,various bacterial growth and expression conditions can be screened,which include different E. Coli cell lines, and growth and proteininduction temperatures. Generally, it is preferred to obtain the maximumamount of soluble protein while still inducing protein expression with arelatively low IPTG concentration e.g., ˜0.2 mM (final concentration) at16° C. Under these conditions, reasonable quantities of protein wereobtained (5-10 mg/ml of soluble protein) using the His-tagged plasmidtransformed in E. Coli BL21 (DE3) cells grown in M9 minimal medium. TheN-terminal His-tag can be readily cleaved with the treatment of proteasethrombin in a 20 mM Tris buffer of pH 8.0 containing 200 mM NaCl and 5mM P-Mercapto-ethanol.

One major advantage of using the heteronuclear multidimensionalapproach, as exemplified herein, is that the NMR resonance assignmentsof a protein are obtained in a sequence-specific manner which assuresaccuracy and greatly facilitates data analysis and structuredetermination [Clore, and Gronenborn, Meth. Enzymol. 239:249-363(1994)]. In addition, the signal overlapping problems in the proteinspectra are minimized by the use of multidimensional NMR spectra, whichseparates the proton signals according to the chemical shifts of theirattached hetero-nuclei (such as ¹⁵N and ¹³C). This NMR approach has beenproven very powerful for structural analysis of large proteins [Cloreand Gronenbom, Meth. Enzymol. 239:249-363 (1994)]. To facilitatesequence-specific resonance assignments for the structural study, auniformly ¹³C, ⁵N-labeled and fractionally (75%) deuterated proteinsample of the SNT-1 PTB domain can be prepared by growing bacterialcells in 75% ²H₂O. Such protein samples can be used for triple-resonanceNMR experiments. A triple-labeled protein sample is useful forhigh-resolution NMR structural studies. Because of the favorable ¹H,¹³C, and ¹⁵N relaxation rates caused by the partial deuteration of theprotein, constant-time triple-resonance NMR spectra can be acquired withhigher digital resolution and sensitivity [Sattler and Fesik, Structure4:1245-1249 (1996)]. In addition, various stable-isotopically labeled(¹⁵N and ¹³C /¹⁵N) proteins can also be prepared using this procedure.

Synthetic Polypeptides

The term “polypeptide” is used in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits are linked by peptide bonds. The FGFRpeptides of the present invention may be chemically synthesized.

In addition, potential drugs or agents that may be tested in the drugscreening assays of the present invention may also be chemicallysynthesized. Synthetic polypeptides, prepared using the well knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (N^(α)-amino protected N^(α)-t-butyloxycarbonyl) amino acidresin with the standard deprotecting, neutralization, coupling and washprotocols of the original solid phase procedure of Merrifield [J. Am.Chem. Soc., 85:2149-2154 (1963)], or the base-labile N^(α)-aminoprotected 9-fluorenylmethoxycarbonyl (Fmoc) amino acids first describedby Carpino and Han [J. Org. Chem., 37:3403-3409 (1972)]. Both Fmoc andBoc N^(α)-amino protected amino acids can be obtained from Fluka,Bachem, Advanced Chemtech, Sigma, Cambridge Research Biochemical,Bachem, or Peninsula Labs or other chemical companies familiar to thosewho practice this art. In addition, the method of the invention can beused with other N^(α)-protecting groups that are familiar to thoseskilled in this art. Solid phase peptide synthesis may be accomplishedby techniques familiar to those in the art and provided, for example, inStewart and Young [Solid Phase Synthesis, Second Edition, PierceChemical Co., Rockford, Ill. (1984)] and Fields and Noble [Int. J. Pept.Protein Res., 35:161-214 (1990)], or using automated synthesizers, suchas sold by ABS. Thus, polypeptides of the invention may comprise D-aminoacids, a combination of D- and L-amino acids, and various “designer”amino acids (e.g., β-methyl amino acids, Cα-methyl amino acids, andNα-methyl amino acids, etc.) to convey special properties. Syntheticamino acids include ornithine for lysine, fluorophenylalanine forphenylalanine, and norleucine for leucine or isoleucine. Additionally,by assigning specific amino acids at specific coupling steps, α-helices,β turns, β sheets, γ-turns, and cyclic peptides can be generated.

In a further embodiment, subunits of peptides that confer usefulchemical and structural properties will be chosen. For example, peptidescomprising D-amino acids will be resistant to L-amino acid-specificproteases in vivo. In addition, the present invention envisionspreparing peptides that have more well defined structural properties,and the use of peptidomimetics, and peptidomimetic bonds, such as esterbonds, to prepare peptides with novel properties. In another embodiment,a peptide may be generated that incorporates a reduced peptide bond,i.e., R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues orsequences. A reduced peptide bond may be introduced as a dipeptidesubunit. Such a molecule would be resistant to peptide bond hydrolysis,e.g., protease activity. Such peptides would provide ligands with uniquefunction and activity, such as extended half-lives in vivo due toresistance to metabolic breakdown, or protease activity. Furthermore, itis well known that in certain systems constrained peptides show enhancedfunctional activity [Hruby, Life Sciences, 31:189-199 (1982); Hruby etal., Biochem J., 268:249-262 (1990)]; the present invention provides amethod to produce a constrained peptide that incorporates randomsequences at all other positions.

Constrained and cyclic peptides. A constrained, cyclic or rigidizedpeptide may be prepared synthetically, provided that in at least twopositions in the sequence of the peptide an amino acid or amino acidanalog is inserted that provides a chemical functional group capable ofcrosslinking to constrain, cyclise or rigidize the peptide aftertreatment to form the crosslink. Cyclization will be favored when aturn-inducing amino acid is incorporated. Examples of amino acidscapable of crosslinking a peptide are cysteine to form disulfides,aspartic acid to form a lactone or a lactam, and a chelator such asγ-carboxyl-glutamic acid (Gla) (Bachem) to chelate a transition metaland form a cross-link. Protected γ-carboxyl glutamic acid may beprepared by modifying the synthesis described by Zee-Cheng and Olson[Biophys. Biochein. Res. Commun., 94:1128-1132(1980)]. A peptide inwhich the peptide sequence comprises at least two amino acids capable ofcrosslinking may be treated, e.g., by oxidation of cysteine residues toform a disulfide or addition of a metal ion to form a chelate, so as tocrosslink the peptide and form a constrained, cyclic or rigidizedpeptide.

The present invention provides strategies to systematically preparecross-links. For example, if four cysteine residues are incorporated inthe peptide sequence, different protecting groups may be used (Hiskey,in The Peptides: Analysis, Synthesis, Biology, Vol. 3, Gross andMeienhofer, eds., Academic Press: New York, pp. 137-167 (1981); Ponsantiet al., Tetrahedron, 46:8255-8266 (1990)]. The first pair of cysteinesmay be deprotected and oxidized, then the second set may be deprotectedand oxidized. In this way a defined set of disulfide cross-links may beformed. Alternatively, a pair of cysteines and a pair of chelating aminoacid analogs may be incorporated so that the cross-links are of adifferent chemical nature.

Non-classical amino acids that induce conformational constraints. Thefollowing non-classical amino acids may be incorporated in the peptidein order to introduce particular conformational motifs:1,2,3,4-tetrahydroisoquinoline-3-carboxylate [Kazmierski et al., J. Am.Chem. Soc., 113:2275-2283 (1991)]; (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, Tetrahedron Lett.(1991)]; 2-aminotetrahydronaphthalene-2-carboxylic acid [Landis, Ph.D.Thesis, University of Arizona (1989)];hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate [Miyake et al. J.Takeda Res. Labs., 43:53-76 (1989)]; β-carboline (D and L) [Kazmierski,Ph.D. Thesis, University of Arizona (1988)]; HIC (histidine isoquinolinecarboxylic acid) [Zechel et al., Int. J. Pep. Protein Res., 43 (1991)];and HIC (histidine cyclic urea) (Dharanipragada).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog [Kemp et al., J. Org. Chem., 50:5834-5838 (1985)];β-sheet inducing analogs [Kemp et al, Tetrahedron Lett., 29:5081-5082(1988); β-turn inducing analogs [Kemp et al., Tetrahedron Lett.,29:5057-5060 (1988)]; ∝-helix inducing analogs (Kemp et al., TetrahedronLett., 29:4935-4938 (1988)]; γ-tum inducing analogs [Kemp et al., J.Org. Chem., 54:109:115 (1989)]; and analogs provided by the followingreferences: Nagai and Sato, Tetrahedron Lett., 26:647-650 (1985); DiMaioet al., J. Chem. Soc. Perkin Trans., p. 1687 (1989); also a Gly-Ala turnanalog [Kahn et al., Tetrahedron Lett., 30:2317 (1989)]; amide bondisostere [Jones et al., Tetrahedron Lett., 29:3853-3856 (1988)];tretrazol [Zabrocki et al., J. Am. Chem. Soc., 110:5875-5880 (1988)];DTC [Samanen et al., Int. J. Protein Pep. Res., 35:501:509 (1990)]; andanalogs taught in Olson et al., J. Am. Chem. Sci., 112:323-333 (1990)and Garvey et al., J. Org. Chem., 56:436 (1990). Conformationallyrestricted mimetics of beta turns and beta bulges, and peptidescontaining them, are described in U.S. Pat. No. 5,440,013, issued Aug.8, 1995 to Kahn.

Structure-Based Mutation Analysis

Protein structural analysis using NMR spectroscopy has several uniqueadvantages. In addition to high-resolution three-dimensional structuralinformation, the chemical shift assignments for the protein obtained inthe structural study further provides a map of the entire protein at theatomic level, which can be used for structure-based biochemical analysisof protein-protein interactions. For example, the information generatedfrom the NMR structural analysis can also serve to identify specificamino acid residues in the peptide-binding site for complementarymutagenesis studies. Specific focus can be placed on those residues thatdisplay long-range NOEs (particularly the side-chain NOEs in the¹³C-NOESY data) between the PTB domain and the FGFR peptides, forexample.

To ensure mutant proteins are valid for functional analysis, it can bedetermined as to whether a mutation results in any significantperturbation of the overall conformation of the PTB domain, particularlythe effects of mutation on the peptide binding sites. NMR spectroscopyis a powerful method for examining the effects of such a mutation on theconformation of the protein. One can readily obtain information aboutthe global conformation of a mutant protein from the proton (¹H) 1Dspectrum, by examining the chemical shift dispersion and peak line-widthof NMR signals of amide, aromatic and aliphatic protons. Moreover, 2D¹H-¹⁵N HSQC spectra reveal details of the effects of a mutation on bothlocal and global conformation of the protein, since every single ¹H/¹⁵Nsignal (both the chemical shift and line-shape) in the NMR spectrum is a“reporter” for a particular amino acid residue. Thus, to assess howmutations effect protein stability and the overall protein conformation,the ¹⁵N HSQC spectra of mutated proteins can be compared to that of thewild-type protein.

Chemical-shift perturbations due to ligand binding have proven to be areliable and sensitive probe for the ligand binding site of the protein.This is because the chemical-shift changes of the backbone amide groupsare likely to reflect any changes in protein conformation and/orhydrogen bonding due to the peptide/ligand binding. To examine theeffects of a mutation on the ligand binding (in this case the ligand isa peptide), peptide titration experiments can be conducted by followingthe changes of ¹H/¹⁵N signals of the mutant proteins as a function ofthe peptide concentration. These experiments indicate whether thepeptide-binding site remains the same or changes in the mutants relativeto the wild type protein. The effects of the mutation on the peptidebinding affinity can also be examined by NMR spectroscopy. Due to thehigh binding affinity of the peptides for the wild type SNT-1 PTBdomain, signals of the free and peptide-bound forms exhibit a slowexchange on the NMR time scale during the peptide titration. If themutated proteins result in the reduction of the binding affinity, achange of the exchange phenomenon between the free and the ligand-boundsignals should be observed in NMR spectrum. If the reduction in bindingaffinity causes the peptide binding to change from a slow exchange rateto a fast exchange rate, on the NMR time scale, then the peptide bindingaffinity can be determined from the NMR titration experiment. From thesemutation analyses key amino acid residues that are important for bindingeither the tyrosine-phosphorylated TRKA receptor peptide or thenon-phosphorylated FGF receptor peptide can be identified.

Protein Structure Determination by NMR Spectroscopy

The NMR results from the present invention are summarized in Table 1,which includes exemplary structural coordinates, Table 2, which containsthe chemical shift data, Table 3, which contains the Hbond results,Table 4, which contains the unambiguous determinations, and Table 5,which contains the ambiguous determinations.

Backbone and Side-chain Assignments: Sequence-specific backboneassignment is achieved by using a suite of deuterium-decoupledtriple-resonance 3D NMR experiments which include HNCA, HN(CO)CA,HN(CA)CB, HN(COCA)CB, HNCO, and HN(CA)CO experiments [Yamazaki, et al.,J. Am. Chem. Soc. 116:11655-11666 (1994)]. The water flip-back scheme isused in these NMR pulse programs to minimize amide signal attenuationfrom water exchange. Sequential side-chain assignments are typicallyaccomplished from a series of 3D NMR experiments with alternativeapproaches to confirm the assignments. These experiments include 3D ¹⁵NTOCSY—HSQC, HCCH-TOCSY, (H)C(CO)NH-TOCSY, and H(C)(CO)NH-TOCSY [seeClore and Gronenborn, Meth. Enzymol. 239:249-363 (1994); Sattler et al.,Prog. in Nuclear Magnetic Resonance Spec. 4:93-158 (1999)].

Stereospecific Methyl Groups: Stereospecific assignments of methylgroups of Valine and Leucine residues are obtained from an analysis ofcarbon signal multiplet splitting using a fractionally ¹³C-labeledprotein sample, which can be readily prepared using M9 minimal mediumcontaining 10% ¹³C-/90% ¹²C-glucose mixture [see Neri, et al.,Biochemistry 28:7510-7516 (1989)].

Dihedral Angle Restraints: Backbone dihedral angle (Φ) constraints aregenerated from the ³J_(HNHα) coupling constants measured in a HNHA-Jexperiment [see Vuister, G. & Bax, A. J. Am. Chem. Soc. 115:7772-7777(1993)]. Side-chain dihedral angles (χ1) can be obtained from shortmixing time ¹⁵N-edited 3D TOCSY—HSQC [see Clore, et al., J. Biomol. NMR1:13-22 (1991)] and 3D HNHB experiments [see Matson et al., J. Biomol.NMR 3:239-244 (1993)], which can also provide stereospecific assignmentsof β methylene protons.

Hydrogen Bonds Restraints: Amide protons that are involved in hydrogenbonds can be identified from an analysis of amide exchange ratesmeasured from a series of 2D ¹H/¹⁵N HSQC spectra recorded after adding²H₂O to the protein sample.

NOE Distance Restraints: Distance restraints are obtained from analysisof ¹⁵N, and ¹³C-edited 3D NOESY data, which can be collected withdifferent mixing times to minimize spin diffusion problems. The nuclearOverhauser effect (NOE)-derived restraints are categorized as strong(1.8-3 Å), medium (1.8-4 Å) or weak (1.8-5 Å) based on the observed NOEintensities. A recently developed procedure for the iterative automatedNOE analysis by using ARIA [see Nilges et al., Prog. NMR Spectroscopy32:107-139 (1998)] can be employed which integrates with X-PLOR forstructural calculations. To ensure the success of ARIA/X-PLOR-assistedNOE analysis and structure calculations, the ARIA assigned NOE peaks canbe manually confirmed.

Intermolecular NOE Distance Restrains: For the structural determinationof a protein/peptide complex, intermolecular NOE distance restraints canbe obtained from a ¹³C-edited (F₁) and ¹⁵N, and ¹³C-filtered (F₃) 3DNOESY data set collected for a sample containing isotope-labeled proteinand non-labeled peptide.

Structure Calculations and Refinements: Structures of the protein can begenerated using a distance geometry/simulated annealing protocol withthe X-PLOR program [see Nilges et al., FEBS Lett. 229:317-324 (1988);Kuszewski, et al., J. Biolmol NMR 2:33-56 (1992); Brunger, X-PLORVersion 3.1: A system for X-Ray crystallography and NMR (Yale UniversityPress, New Haven, Conn., 1993)]. The structure calculations can employinter-proton distance restraints obtained from ¹⁵N— and ¹³C-resolvedNOESY spectra. The initial low-resolution structures can be used tofacilitate NOE assignments, and help identify hydrogen bonding partnersfor slowly exchanging amide protons. The experimental restraints ofdihedral angles and hydrogen bonds can be included in the distancerestraints for structure refinements.

Protein-Structure Based Design of Agonists and Antagonists of TheSNT/FGFR Complex

Once the three-dimensional structure of the SNT/FGFR complex isdetermined, a potential drug or agent (antagonist or agonist) can beexamined through the use of computer modeling using a docking programsuch as GRAM, DOCK, or AUTODOCK [Dunbrack et al., 1997, supra]. Thisprocedure can include computer fitting of potential agents to the PTBdomain of SNT-1, for example, to ascertain how well the shape and thechemical structure of the potential ligand will complement or interferewith the interaction between SNT-1 and FGFR [Bugg et al., ScientificAmerican, December:92-98 (1993); West et al., TIPS, 16:67-74 (1995)].Computer programs can also be employed to estimate the attraction,repulsion, and steric hindrance of the agent to the dimer-dimer bindingsite, for example. Generally the tighter the fit (e.g., the lower thesteric hindrance, and/or the greater the attractive force) the morepotent the potential drug will be since these properties are consistentwith a tighter binding constant. Furthermore, the more specificity inthe design of a potential drug the more likely that the drug will notinterfere with related proteins. This will minimize potentialside-effects due to unwanted interactions with other proteins.

Initially a potential drug could be obtained by screening a randompeptide library produced by recombinant bacteriophage for example,[Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc.Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science,249:404-406 (1990)] or a chemical library. An agent selected in thismanner could be then be systematically modified by computer modelingprograms until one or more promising potential drugs are identified.Such analysis has been shown to be effective in the development of HIVprotease inhibitors [Lam et al., Science 263:380-384 (1994); Wlodawer etal., Ann. Rev. Biochem. 62:543-585. (1993); Appelt, Perspectives in DrugDiscovery and Design 1:23-48 (1993); Erickson, Perspectives in DrugDiscovery and Design 1:1-09-128 (1993)].

Such computer modeling allows the selection of a finite number ofrational chemical modifications, as opposed to the countless number ofessentially random chemical modifications that could be made, any ofwhich any one might lead to a useful drug. Each chemical modificationrequires additional chemical steps, which while being reasonable for thesynthesis of a finite number of compounds, quickly becomes overwhelmingif all possible modifications needed to be synthesized. Thus through theuse of the three-dimensional structural analysis disclosed herein andcomputer modeling, a large number of these compounds can be rapidlyscreened on the computer monitor screen, and a few likely candidates canbe determined without the laborious synthesis of untold numbers ofcompounds.

Once a potential drug (agonist or antagonist) is Identified it can beeither selected from a library of chemicals as are commerciallyavailable from most large chemical companies including Merck,GlaxoWelcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly, Novartisand Pharmacia Upjohn, or alternatively the potential drug may besynthesized de novo. As mentioned above, the de novo synthesis of one oreven a relatively small group of specific compounds is reasonable in theart of drug design.

The potential drug can then be tested in any standard binding assay(including in high throughput binding assays) for its ability to bind toSNT or fragment thereof comprising the PTB domain. Alternatively thepotential drug can be tested for its ability to modulate (either inhibitor stimulate) a cellular signal that is dependent on the interaction ofSNT with FGFR. When a suitable potential drug is identified, a secondNMR structural analysis can optionally be performed on the bindingcomplex formed between the SNT/FGFR complex and the potential drug.Computer programs that can be used to aid in solving suchthree-dimensional structures include QUANTA, CHARMM, INSIGHT, SYBYL,MACROMODE, and ICM, MOLMOL, RASMOL, AND GRASP [Kraulis, J. ApplCrystallogr. 24:946-950 (1991)]. Most if not all of these programs andothers as well can be also obtained from the WorldWideWeb through theinternet.

Using the approach described herein and equipped with the structuralanalysis disclosed herein, the three-dimensional structures of otherSNT/receptor complexes can more readily be obtained and analyzed. Suchanalysis will, in turn, allow corresponding drug screening methodologyto be performed using the three-dimensional structures of such relatedcomplexes.

For all of the drug screening assays described herein furtherrefinements to the structure of the drug will generally be necessary andcan be made by the successive iterations of any and/or all of the stepsprovided by the particular drug screening assay, including furtherstructural analysis by NMR, for example.

Phage Libraries for Drug Screening.

Phage libraries have been constructed which when infected into host E.coli produce random peptide sequences of approximately 10 to 15 aminoacids [Parmley and Smith, Gene 73:305-318 (1988), Scott and Smith,Science 249:386-249 (1990)]. Specifically, the phage library can bemixed in low dilutions with permissive E. coli in low melting point LBagar which is then poured on top of LB agar plates. After incubating theplates at 37° C. for a period of time, small clear plaques in a lawn ofE. coli will form which represents active phage growth and lysis of theE. coli. A representative of these phages can be absorbed to nylonfilters by placing dry filters onto the agar plates. The filters can bemarked for orientation, removed, and placed in washing solutions toblock any remaining absorbent sites. The filters can then be placed in asolution containing, for example, a radioactive PTB domain (e.g., thepeptide comprising amino acid residues 11-140 of SEQ ID NO:1). After aspecified incubation period, the filters can be thoroughly washed anddeveloped for autoradiography. Plaques containing the phage that bind tothe radioactive PTB domain can then be identified. These phages can befurther cloned and then retested for their ability to bind to the PTBdomain as before. Once the phage has been purified, the binding sequencecontained within the phage can be determined by standard DNA sequencingtechniques. Once the DNA sequence is known, synthetic peptides can begenerated which are encoded by these sequences.

These peptides can be tested, for example, for their ability to e.g.,interfere with the binding of SNT with FGFR.

The effective peptide(s) can be synthesized in large quantities for usein in vivo models and eventually in humans to treat certain tumors. Itshould be emphasized that synthetic peptide production is relativelynon-labor intensive, easily manufactured, quality controlled and thus,large quantities of the desired product can be produced quite cheaply.Similar combinations of mass produced synthetic peptides have recentlybeen used with great success [Patarroyo, Vaccine, 10:175-178 (1990)].

Drug Screening Assays

The drug screening assays of the present invention may use any of anumber of means for determining the interaction between an agent or drugand SNT and/or FGFR. In one such assay, a drug can be specificallydesigned to bind to the PTB domain of SNT-1 through NMR basedmethodology. [Shuker et al., Science 274:1531-1534 (1996) herebyincorporated by reference in its entirety.] In a particular embodiment,analogs of the FGFR derived peptide having the amino acid sequence ofSEQ ID NO:3 are used. In a particular embodiment of this type, thepeptide has the amino acid sequence of SEQ ID NO:4. In another suchembodiment of this type, the peptide has the amino acid sequence of SEQID NO:5. Alternatively, a library of low molecular weight compounds canbe screened to identify a binding partner for the PTB domain. Any suchchemical library can be used including those discussed above.

The assay: begins with contacting a compound with a ¹⁵N-labeled SNT PTBdomain. Binding of the compound with the SNT PTB domain can bedetermined by monitoring the ¹⁵N— or ¹H-amide chemical shift changes intwo dimensional ¹⁵N-heteronuclear single-quantum correlation (¹⁵N—HSQC)spectra upon the addition of the compound to the ¹⁵N-labeled SNT PTBdomain. Since these spectra can be rapidly obtained, it is feasible toscreen a large number of compounds [Shuker et al., Science 274:1531-1534(1996)]. A compound is identified as a potential ligand if it binds tothe SNT PTB domain. In a further embodiment, the potential ligand canthen be used as a model structure, and analogs to the compound can beobtained (e.g., from the vast chemical libraries commercially available,or alternatively through de novo synthesis). The analogs are thenscreened for their ability to bind the SNT PTB domain to obtain aligand. An analog of the potential ligand is chosen as a ligand when itbinds to the SNT PTB domain with a higher binding affinity than thepotential ligand. In a preferred embodiment of this type the analogs arescreened by monitoring the ¹⁵N— or ¹H-amide chemical shift changes intwo dimensional ¹⁵N-heteronuclear single-quantum correlation (¹⁵N—HSQC)spectra upon the addition of the analog to the ¹⁵N-labeled SNT domain asdescribed above.

In another further embodiment, compounds are screened for binding to twonearby sites on an SNT PTB domain. In this case, a compound that binds afirst site of the SNT PTB domain does not bind a second nearby site.Binding to the second site can be determined by monitoring changes in adifferent set of amide chemical shifts in either the original screen ora second screen conducted in the presence of a ligand (or potentialligand) for the first site. From an analysis of the chemical shiftchanges the approximate location of a potential ligand for the secondsite is identified. Optimization of the second ligand for binding to thesite is then carried out by screening structurally related compounds(e.g., analogs as described above). When ligands for the first site andthe second site are identified, their location and orientation in theternary complex can be determined experimentally either by NMRspectroscopy or X-ray crystallography. On the basis of this structuralinformation, a linked compound is synthesized in which the ligand forthe first site and the ligand for the second site are linked. In apreferred embodiment of this type the two ligands are covalently linked.This linked compound is tested to determine if it has a higher bindingaffinity for the SNT PTB domain than either of the two indiviualligands. A linked compound is selected as a ligand when it has a higherbinding affinity for the SNT PTB domain than either of the two ligands.In a preferred embodiment the affinity of the linked compound with theSNT PTB domain is determined monitoring the ¹⁵N— or ¹H-amide chemicalshift changes in two dimensional ¹⁵N-heteronuclear single-quantumcorrelation (¹⁵N—HSQC) spectra upon the addition of the linked compoundto the ¹⁵N-labeled SNT PTB domain as described above.

A larger linked compound can be constructed in an analogous manner,e.g., linking three ligands which bind to three nearby sites on the SNTPTB domain to form a multilinked compound that has an even higheraffinity for the SNT PTB domain than linked compound.

In another assay, a SNT PTB domain is placed on or coated onto a solidsupport. Methods for placing the peptides or proteins on the solidsupport are well known in the art and include such things as linkingbiotin to the protein and linking avidin to the solid support. An agentis allowed to equilibrate with the SNT PTB domain to test for binding.Generally, the solid support is washed and agents that are retained areselected as potential drugs. In a particular embodiment of this type,the SNT PTB domain comprises amino acid residues 11-140 of SEQ ID NO:1.

The agent may be labeled. For example, in one embodiment radiolabeledagents are used to measure the binding of the agent. In anotherembodiment the agents have fluorescent markers. In yet anotherembodiment, a Biocore chip (Pharmacia) coated with the SNT PTB domain isused and the change in surface conductivity can be measured.

In yet another embodiment, the affect of a prospective drug (a testcompound or agent) on a SNT PTB domain is assayed in a living cell thatcontains or can be induced to contain SNT-1 and FGFR. The cell can alsocontain or can be constructed to contain one or more reporter genes,such as a heterologous gene comprising a nucleic acid encodingluciferase, green fluorescent protein, chloramphenicol acetyltransferase, and/or β-galactosidase etc. Such reporter genes can beoperably linked to a promoter comprising a binding site for atranscription factor under the control of the SNT/FGFR dependentcellular signaling pathway. Cells that naturally encode a FGFR and SNT-1may be used, or alternatively a cell that is transfected with plasmidsencoding the reporter proteins can be used, though care must be taken toensure that the requisite participants in the SNT/FGFR dependentcellular signaling pathway are also present.

Assays for detecting the reporter genes products are readily availablein the literature. For example, luciferase assays can be performedaccording to the manufacturer's protocol (Promega), and β-galactosidaseassays can be performed as described by Ausubel et al. [in CurrentProtocols in Molecular Biology, J. Wiley & Sons, Inc. (1994)]. Thepreparation of such plasmid containing reporter genes is now routine inthe art, and many appropriate plasmids are now commercially availablewhich can be readily modified for such assays.

The prospective drug is generally tested under conditions in which theFGFR has been activated. The FGF receptor can be activated by anantibody or preferably FGF. Alternatively, a permantively activated FGFcan be constitutively expressed. In one embodiment, the expression ofSNT-1 is constitutive. The amount (and/or activity) of reporter proteinproduced in the absence and presence of prospective drug can then bedetermined and compared. Prospective drugs which reduce the amount(and/or activity) of the reporter protein produced are candidateantagonists of the SNT/FGFR complex, whereas prospective drugs whichincrease the amount (and/or activity) of reporter protein produced arecandidate agonists.

Labels

Suitable labels include enzymes, fluorophores (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine,free or chelated lanthanide series salts, especially Eu³⁺; to name a fewfluorophores), chromophores, radioisotopes, chelating agents, dyes,colloidal gold, latex particles, ligands (e.g., biotin), andchemiluminescent agents. When a control marker is employed, the same ordifferent labels may be used for the test and control marker gene.

In the instance where a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, 35S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re areused, known currently available counting procedures may be utilized. Inthe instance where the label is an enzyme, detection may be accomplishedby any of the presently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

Direct labels are one example of labels which can be used according tothe present invention. A direct label has been defined as an entity,which in its natural state, is readily visible, either to the naked eye,or with the aid of an optical filter and/or applied stimulation, e.g,ultraviolet light to promote fluorescence. Among examples of coloredlabels, which can be used according to the present invention, includemetallic sol particles, for example, gold sol particles such as thosedescribed by Leuvering (U.S. Pat. No. 4,313,734); dye sole particlessuch as described by Gribnau et al. (U.S. Pat. No. 4,373,932 and May etal. (WO 88/08534); dyed latex such as described by May, supra, Snyder(EP-A 0 280 559 and 0 281 327); or dyes encapsulated in liposomes asdescribed by Campbell et al. (U.S. Pat. No. 4,703,017). Other directlabels include a radionucleotide, a fluorescent moiety or a luminescentmoiety. In addition to these direct labeling devices, indirect labelscomprising enzymes can also be used according to the present invention.Various types of enzyme linked immunoassays are well known in the art,for example, alkaline phosphatase and horseradish peroxidase, lysozyme,glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease, theseand others have been discussed in detail by Eva Engvall in EnzymeImmunoassay ELISA and EMIT in Methods in Enzymology, 70:419-439 (1980)and in U.S. Pat. No. 4,857,453.

Suitable enzymes include, but are not limited to, alkaline phosphatase,β-galactosidase, green fluorescent protein and its derivatives,luciferase, and horseradish peroxidase.

Other labels for use in the invention include magnetic beads or magneticresonance imaging labels.

Antibodies to the SNT and FGFR Fragments

According to the present invention, the SNT and FGFR peptides andfragments as produced by a recombinant source, or through chemicalsynthesis, or through the modification of these peptides and fragments;and derivatives or analogs thereof, including fusion proteins, may beused as an immunogen to generate antibodies that specifically interferewith the formation of the SNT/FGFR complex. Such antibodies include butare not limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, and a Fab expression library.

Various procedures known in the art may be used for the production ofthe polyclonal antibodies. For the production of antibody, various hostanimals can be immunized by injection with the peptide having the aminoacid sequence of SEQ ID NO:3 for example, or a derivative (e.g., afusion protein) thereof, including but not limited to rabbits, mice,rats, sheep, goats, etc. In one embodiment, the peptide can beconjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA)or keyhole limpet hemocyanin (KLH). Various adjuvants may be used toincrease the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies directed toward the peptides orprotein fragments of the present invention, or analog, or derivativethereof, any technique that provides for the production of antibodymolecules by continuous cell lines in culture may be used. These includebut are not limited to the hybridoma technique originally developed byKohler and Milstein [Nature, 256:495-497 (1975)], as well as the triomatechnique, the human B-cell hybridoma technique [Kozbor et al.,Immunology Today, 4:12 (1983); Cote et al., Proc. Natl. Acad. Sci.U.S.A., 80:2026-2030 (1983)], and the EBV-hybridoma technique to producehuman monoclonal antibodies [Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology [PCT/US90/02545]. In fact,according to the invention, techniques developed for the production of“chimeric antibodies” [Morrison et al., J. Bacteriol., 159:870 (1984);Neuberger et al., Nature, 312:604-608 (1984); Takeda et al., Nature,314:452-454 (1985)] by splicing the genes from a mouse antibody moleculespecific for the peptide having the amino acid sequence of SEQ ID NO:3together with genes from a human antibody molecule of appropriatebiological activity can be used; such antibodies are within the scope ofthis invention. Such human or humanized chimeric antibodies arepreferred for use in therapy of human diseases or disorders (describedinfra), since the human or humanized antibodies are much less likelythan xenogenic antibodies to induce an immune response, in particular anallergic response, themselves.

According to the invention, techniques described for the production ofsingle chain antibodies [U.S. Pat. Nos. 5,476,786 and 5,132,405 toHuston; U.S. Pat. No. 4,946,778] can be adapted to produce specificsingle chain antibodies. An additional embodiment of the inventionutilizes the techniques described for the construction of Fab expressionlibraries [Huse et al., Science, 246:1275-1281 (1989)] to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immununodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of an FGFR, for example, one may assay generatedhybridomas for a product which binds to an FGFR fragment containing suchepitope and choose those which do not cross-react with FGFR fragmentsthat do not include that epitope.

In a specific embodiment, antibodies that interfere with the formationof the SNT/FGFR complex can be generated. Such antibodies can be testedusing the assays described could potentially be used in anti-tumortherapies.

The present invention may be better understood by reference to thefollowing non-limiting Example, which is provided as exemplary of theinvention. The following example is presented in order to more fullyillustrate the preferred embodiments of the invention. It should in noway be construed, however, as limiting the broad scope of the invention.

EXAMPLE The Three-Dimensional Structure of the SNT-1 PTB Domain With theFGF Receptor Introduction

Phosphotyrosine binding (PTB) domains represent a group of structurallyrelated but functionally divergent protein modules that play animportant role in regulating protein-phosphotyrosine,protein-phospholipids, or protein-protein interactions [Pawson, andScott, Nature 278: 2075-2080 (1997); Zhou and Fesik, Prog. Biophys.Molec. Biol. 64:221-235 (1995)]. The amino-terminal PTB domains of thenewly discovered lipid-anchored docking proteins SNT-1 and SNT-2 havebeen shown to possess a unique ability to recognize both a canonicalNPXpY motif on the activated nerve growth factor (NGF) receptor, and atyrosine and asparagine-free motif on the fibroblast growth factor (FGF)receptor [Xu et al., J. Biol. Chem. 273:17987-17990 (1998); Kouhara etal. Cell 89:693-702 (1997); Meakin et al., J. Biol. Chem. 274:9861-9870(1999)].

Materials and Methods

Sample Preparation: A cDNA fragment encoding the SNT-1 PTB domain wascloned into a modified pET28b vector (Novagen) that produced therecombinant protein with a cleavable hexa-histidine (His₆) tag at theC-terminus. Uniformly ¹⁵N— and ¹⁵N/¹³C-labeled proteins were prepared bygrowing Escherichia coli BL21(DE3) cells in a minimal medium containing¹⁵NH₄Cl with or without ¹³C₆-glucose. A uniformly ¹⁵N/¹³C-labeled andfractionally deuterated protein was prepared using medium with 75% ²H₂O.The protein was over-expressed largely in soluble form and purified byaffinity chromatography on a nickel-IDA column (Invitrogen) followed bycleavage of the His₆ tag upon thrombin treatment. The cleaved proteincontains an additional LVPR sequence at the C-terminus from theengineered thrombin site. The protein was unstable in its free form andquickly aggregated or became partially unfolded at room temperature. Toensure structural integrity, the protein was further subjected to aprotein refolding procedure followed by ion-exchange chromatography.Synthetic peptides were prepared on a MilliGen 9050 peptide synthesizer(Perkin Elmer) using Fmoc/HBTU chemistry. NMR samples contained theSNT-1 PTB domain/hFGFR1 peptide complex (1:1) of 0.5 mM in 100 mMphosphate buffer of pH 6.5, 5 mM DTT-d₁₀ and 0.5 mM EDTA in H₂O/²H₂O(9/1) or ²H₂O.

NMR Spectroscopy: NMR spectra were acquired at 30° C. on a Bruker DRX600or DRX500 spectrometer. The backbone and side-chain ¹H, ¹³C and ¹⁵Nresonances of the protein were assigned using deuterium-decoupledtriple-resonance experiments of HNCA, HN(CO)CA, HNCACB, HN(CO)CACB and(H)C(CO)NH-TOCSY [Yamazaki, et al., J. Am. Chem. Soc. 116:11655-11666(1994); Sattler et al., Prog. in Nuclear Magnetic Resonance Spec.4:93-158 (1999)] recorded using uniformly ¹⁵N/¹³C-labeled andfractionally deuterated protein in complex with a non-isotopicallylabeled hFGFR1 peptide. The side-chain assignments were completed using3D HCCH-TOCSY [Clore, and Gronenbom, Meth. Enzymol. 239:249-363 (1.994)]data collected from a uniformly ¹⁵N/¹³C-labeled protein/non-labeledpeptide complex NOE-derived distance restraints were obtained from ¹⁵N—or ¹³C-edited 3D NOESY spectra [Clore, and Gronenborn, Meth. Enzymol.239:249-363 (1994)]. φ angle restraints were determined from ³J_(HN,Hα)coupling constants measured in a 3D HNHA-J spectrum [Clore, andGronenborn, Meth. Enzymol. 239:249-363 (1994)]. Slowly exchanging amideprotons were identified from a series of 2D ¹⁵N—HSQC spectra recordedafter the H₂O buffer was changed to ^(2H) ₂O buffer. The peptideresonances were assigned using ¹³C/¹⁵N-filtered 2D NOESY and TOCSYspectra [Sattler et al., Prog. in Nuclear Magnetic Resonance Spec.4:93-158 (1999)] collected from a ¹⁵N/¹³C-labeled protein/non-labeledpeptide complex. The intermolecular NOEs used in defining the structureof the SNT-1 PTB domain/hFGFR1 complex were detected in ¹³C— or¹⁵N-edited (F₁), ¹³C/¹⁵N-filtered (F₃) 3D NOESY spectra. All NMR spectrawere processed with NMRPipe/NMRDraw [Delaglio et al., J. Biomol. NMR6:277-293 (1995)] and analyzed using NMRView [Johnson and Blevins J.Biomol. NMR 4:603-614 (1994)]. Chemical shift assignments of the SNT PTBdomain and the hFGFR1 peptide have been deposited in the BioMagResBank(BMRB) under accession number 4790.

Structure Calculations: Structures of the SNT-1 PTB domain in complexwith the hFGFR1 peptide were calculated with a distance geometry andsimulated annealing protocol using the X-PLOR program [Brunger, X-PLORVersion 3.1: A system for X-Ray crystallography and NMR (Yale UniversityPress, New Haven, Conn., 1993)]. NOE distance and dihedral anglerestraints were treated with a square-well potential of 50 kcal mol⁻¹ÅA⁻². A total of 2448 manually assigned NOE-derived distance restraintswere obtained from the ¹⁵N— or ¹³C-edited NOESY data, including 251intra-peptide and 258 intermolecular distance restraints. Additionally,255 unambiguous and 52 ambiguous distance restraints were identifiedfrom the NOE data by using ARIA [Nilges and O'Donoghue, Prog. NMRSpectroscopy 32:107-139 (1998)]. The final structure calculationsemployed a total of 2755 NOE restraints obtained from the manual and theARIA-assisted assignments, 2703 of which were unambiguously assignedNOE-derived distance restraints that comprise of 1072 intra-residue, 466sequential, 216 medium-range and 949 long-range NOEs. In addition, 70hydrogen-bond distance restraints for 35 hydrogen bonds and 19 (φ-anglerestraints were also used in the structure calculations. For theensemble of the final 20 structures, no distance or torsional anglerestraint was violated by more than 0.4 Å or 5°, respectively. Thetotal, distance violation and dihedral violation energies were 262.0±6.0kcal mol⁻¹, 74.4±1.7 kcal mol⁻¹ and 0.82±0.08 kcal mol⁻¹, respectively.The Lennard-Jones potential, which was not used during any refinementstage, was −659.3±23.1 kcal mol⁻¹ for the final structures. Ramachandranplot analysis by Procheck-NMR [Laskowski et al., J. Biomol. NMR8:477-486 (1996)] showed that in the final structures of the complex,98.1 % of the backbone geometries of the non-Gly and non-Pro residues inthe complex (protein residues 18-116 and peptide residues 412-430), andnearly 100% in the secondary structure (protein residues 19-24, 35-40,45-49, 52-57, 63-68, 71-76, 85-90, 94-107 and 111-115; and peptideresidues 426-430) lie within energetically favorable or allowed regions.

Mutagenesis and Yeast Two-Hybrid Binding Assays: The yeast two-hybridbinding studies of the SNT-1 PTB domain binding to hFGFR1 ortyrosine-phosphorylated TRK were performed as described previously [Xuet al., J. Biol. Chem. 273:17987-17990 (1998)]. Briefly, SNT-1 cDNAfragments were cloned into the pACT2 expression vector (Clontech) forexpression as GAL4 DNA activation domain (AD) fusion proteins followedby C-terminal AU1-epitope tags. The juxtamembrane region of hFGFR1 wascloned into the pAS2-1 expression vector (Clontech) for expression as aGAL4 DNA binding domain (BD) fusion protein. This plasmid served as atemplate for site-directed mutagenesis of hFGFR1 using the QuikChangekit (Stratagene). DNA sequencing confirmed the mutations. AD and BDplasmids were co-transformed into Saccharomyces cerevisiae strainpJ69-4A and plated onto selective media. The synthetic medium lackingthe amino acids Leu and Trp (Leu⁻, Trp⁻) selected for plasmid uptake.Medium lacking His, Leu and Trp (His⁻, Leu⁻, Trp⁻) but containing 3 mM3-aminotriazole was used to select for interaction of the AD and BDfusion proteins. Levels of protein interaction were scored by relativecolony growth on these plates. Expression of the SNT-1 protein wasconfirmed by immunoprecipitation from yeast lysates using an anti-AU1monoclonal antibody (BAbCo). Western blotting was performed using ananti-AD antibody (Santa Cruz Biotech), goat anti-mouse IgG conjugatedwith horseradish-peroxidase, and developed by chemiluminescence.Similarly, expression of wild type and mutant hFGFR1 was detected byimmunoprecipitation with a rabbit polyclonal antibody specific for BD(Santa Cruz Biotech) and western blotting with mouse monoclonal anti-BDantibody (Santa Cruz Biotech).

Ligand titration. Ligand titration experiments were performed byrecording a series of 2D ¹⁵N— and ¹³C-HSQC spectra on the uniformly¹⁵N—, and ¹⁵N/¹³C-labeled SNT-1 PTB domain (˜0.3 mM), respectively, inthe presence of different amounts of the peptide ligands concentrationranging from 0 to ˜2.0 mM. The protein sample and the stock solutions ofthe ligands were all prepared in the same aqueous buffer containing 100mM phosphate and 5 mM perdeuterated DTT at pH 6.5.

Summary

In an effort to understand the detailed molecular mechanisms by whichSNTs regulate FGF and NGF receptor signaling, the three-dimensionalstructure of the SNT-1 PTB domain in complex with a peptide derived fromthe known SNT-1 binding site on the FGF receptor has been determinedusing NMR spectroscopy. On the basis of the new structural information,key amino acid residues are disclosed that are important for the SNT-1PTB domain interactions with the TRKA and FGF receptors.

To examine the SNT-1 PTB domain binding to the peptides derived fromtyrosine-phosphorylated TRKA and non-phosphorylated FGF receptors, thereceptor peptides were titrated into the protein solution and thechemical shift perturbations of the protein backbone amide resonances inthe HSQC spectra were monitored. The salient inferences emerged from theNMR titration data are that the FGF and: TRKA receptor peptides bind tothe PTB domain in specific but distinct manners. This conclusion issupported by the observation that distinct sets of amino acid residuesundergo chemical shift perturbation upon binding to the differentpeptides in the NMR titration (FIG. 3B). In addition, binding to theFGFR peptide results in more residues to undergo chemical shift changesthan the TRKA binding. Taken together, these results clearly indicatethat the SNT-1 PTB domain employs two different mechanisms in itsinteractions with the FGF and TRKA receptor peptides.

The human suc-1-associated neurotrophic (SNT) factor target, GenBankAccession No. 5730057, has the amino acid sequence of SEQ ID NO:1. Themouse (Mus musculus) fibroblast growth factor receptor-1, GenBankAccession No. AAC52182, has the amino acid sequence of SEQ ID NO:2. Theprotein fragments employed in the NMR structural determination(summarized in Tables 1-5) were the PTB Domain of SNT-1 comprising aminoacid residues 11-140 of SEQ ID NO:1, and a small fragment of FGFR1comprising amino acid residues 409-430 of SEQ ID NO:2, [HSQMA VHKLAKSIPL RRQVT VS (SEQ ID NO:3)]. The NMR spectra were carried out at aconcentration of 0.5 mM SNT-1/FGFR1 in a 1:1 complex in a 0.1 M sodiumphosphate buffer pH 6.5 containing 5 mM DTT and 1 mM EDTA. The resultsof the NMR analysis are included below in Tables 1-5. The depiction ofthe structure is shown in FIG. 2.

Through the use of the information disclosed herein chemical compoundsthat can inhibit or block the SNT-1 PTB domain interaction with FGFreceptor or the NGF TRKA receptor have been designed and examined. Onesuch compound is a tyrosine-phosphorylated peptide having the amino acidsequence of LVIAGNPApYRS, SEQ ID NO:4 (where pY stands forphosphotyrosine). This peptide can bind to the SNT-1 PTB domain withhigh affinity of K_(d)˜1 μM. The affinity of the SNT-1 PTB domainbinding for this peptide is about 10-fold higher than that for the FGFRderived peptide having the amino acid sequence of SEQ ID NO:3.Furthermore, this peptide partially overlaps with the binding of theFGFR peptide having the amino acid sequence of SEQ ID NO:3 on the SNT-1PTB domain. These results suggest that this tyrosine-phosphorylatedpeptide could act as a competitive inhibitor to block the interactionbetween the SNT-1 PTB domain and FGFR, and thus could serve as basis forfurther development of small molecule mimetics that inhibit thisinteraction in cells.

The present study has also identified several key amino acid residues inboth the SNT-1 PTB domain and FGFR1 that are important for the SNT-1 PTBdomain interactions with the FGF receptor or the NGF TRKA receptor. Thefollowing amino acid residues were identified in the SNT-1 PTB domain ofSNT-1 (having the amino acid sequence of SEQ ID NO:1): Asp 28, Asp 29,Phe 89, Val 112, Glu 114, and Glu 119

In the SNT-1 PTB binding domain of FGFR1 of the FGF receptor having theamino acid sequence of SEQ ID NO:2 the following amino acid residueswere identified: Val 414, Leu 417, Ile 421, Leu 423, Arg 425, Val 427,and Val 429.

Results

Structure of the SNT PTB Domain: Nuclear magnetic resonance (NMR)studies were conducted using a 1:1 complex of SNT-1 PTB domain (residues11-140 of SEQ ID NO:1.) and a 22-residue peptide derived from thejuxtamembrane region of hFGFR1 (residues 409-430 of SEQ ID NO:2) (FIGS.1A and 1B). The dissociation constant (K_(D)) of the protein/peptidecomplex was estimated to be ˜10 μM using the isothermal titrationcalorimetry (ITC) technique. This result is consistent with theinteraction being in slow to intermediate exchange on the NMR timescalein NMR titration experiments. The well-defined structure of this complexwas determined from a total of 2844 NMR-derived restraints (FIG. 2A).The protein structure consists of a β-sandwich containing two nearlyorthogonal, antiparallel β-sheets capped at one end by an amphipathicα-helix (FIGS. 2B and 2C), as anticipated from a classical PTB domain.The SNT-1 structure, however, possesses several unforeseen features thatare unique for the conserved PTB domain scaffold. First, unlike allother known PTB domain structures that end with a C-terminal α-helix[Forman-Kay and Pawson, Curr. Opin. Struct. Biol. 9:690-695 (1999)], theSNT-1 PTB domain has an additional β-strand (β8) extending from itsC-terminal α-helix (α1) that molds the hFGFR1 peptide into the secondantiparallel β-sheet (see below). Second, boundaries of secondarystructure elements between SNT and IRS PTB domains do not necessarilycoincide with their amino acid conservation, such as the conserved VEEmotif of β8 (residues 113-115), suggesting that tertiary interactionsare very important in defining structure elements. Third, a C-terminalportion of the SNT-1 construct (residues 116-136) (FIG. 1A), which ishighly homologous (˜45% identity) to al in the IRS-1 PTB domain, islargely structurally disordered. The loss of helical conformation isperhaps due to the presence of Pro residues and change of amphipathicnature of the sequence, which could disrupt helical propensity and alterinteractions with other parts of the protein, respectively. Whilereasons for the conformational discrepancy between these homologoussequences are evident from structural analysis, the functionalimplications of their evolutionary relationship are not clear. Finally,the sequence comprising residues 94-107 in SNT-1, predicted to be alarge insert from sequence homology alignment with IRS proteins,actually forms an α-helix (α1) that blocks one side of the β-sandwich.Together, these unique structural features of the SNT-1 PTB domain mayconfer its distinct function.

Interactions between hFGFR1 Peptide and the SNTPTB Domain: The hFGFR1peptide wraps around the protein molecule with an unusual backboneconformation containing two nearly 90° turns that are orientedorthogonal to each other (FIGS. 2B and 2C). The peptide interactsextensively with the protein by clasping both sides of the β-sandwich(FIG. 2D). The estimated surface area of SNT-1 buried by the boundpeptide is ˜2025 Å², with 18 of the 22 peptide residues displayingintermolecular NOEs to many protein residues (FIG. 1B). The C-terminalQVTVS segment of the peptide (residues 426-430) adopts an antiparallelβ-strand (β′) sandwiched between β5 and β8. Two intermolecular hydrogenbonds bridging β′ and β5, and a large number of NOEs characteristic ofthe antiparallel β-sheet are observed between backbone atoms of thecomplex (FIG. 4A). Side-chains of Val-427 and Val-429 interactextensively with Leu-62, Tyr-65, Phe-98, Met-105, Ile-110 and Val-112 ina hydrophobic core formed between β5 and α1 (FIG. 4B). The peptidefastens onto the other side of the β-sandwich by embedding itsN-terminal MAVH segment (residues 412-415) into a large hydrophobiccavity bounded by the three loops connecting β1/β2, β3/β4 and β16/β7. Inparticular, methyl groups of Val-414 are completely immersed in anaromatic pool of Trp-57, Phe-74, Phe-87 and Phe-89, and also contactLeu-47, Vat-55 and Thr-82 (FIG. 4C). Moreover, Leu-417, Ile-421 andLeu-423 located in the center of the peptide bind to otherwisesolvent-exposed hydrophobic residues Leu-71, Ile-86 and Ala-88 on thesurface of the second β-sheet (FIG. 4D). In addition to hydrophobicinteractions, complementary electrostatic interactions are observed,largely localized at the two turns in the peptide. At one turn, Arg-424pairs with Asp-68, while Arg-425 interacts with Glu-114 and Glu-119. Atthe other turn, Lys-416 and Lys-419 show interactions with a contiguouspatch of three solvent-accessible residues Asp-27, Asp-28 and Asp-29(FIG. 4E). These results indicate that both hydrophobic andelectrostatic interactions are important for SNT-1 and hFGFR1recognition.

To determine the relative contributions of specific hFGFR1 residues toSNT-1 recognition, site-directed mutagenesis was used to alter thepeptide residues that show intermolecular NOEs to the protein (FIG. 1B).The resulting hFGFR1 mutants were analyzed for SNT-1 interaction byyeast two-hybrid binding assays. Substitution of Ala for either Leu-423or Val-429 completely eliminated peptide binding to the SNT-1 PTBdomain, while mutation of Val-414, Leu-417, Arg-425 or Val-427 to Alasignificantly reduced binding (FIG. 6A). The reduced protein-proteininteractions of the individual FGFR1 mutants were not likely due tovariations of protein expression in the yeast cells (FIG. 6A).Particularly, expression levels of the Leu-423-Ala and Val-429-Alamutants were at least as good as that of the wild type. Thesemutagenesis data agree with (1) the observed intermolecular NOEs (FIG.1B); (2) the calculated solvent accessible surface area (particularlyfor the peptide hydrophobic residues); and (3) amino acid conservationof the juxtamembrane region of the FGFR family (FIG. 1B). In addition,these data are also consistent with results of mutational analysis ofFGFR1 and SNT-1 interactions [Ong et al., Mol. Cell. Biol. 20:979-989(2000)]. Collectively, these results demonstrate that the nature of theprotein and peptide binding is highly specific and extensive, withmultiple types of interactions stabilizing the complex. It isinteresting to note that utilization of the hydrophobic side of theβ-sandwich opposite to α1 for protein interactions in SNT-1 is atypicaland not seen in other PTB domains [Forman-Kay and Pawson, Curr. Opin.Struct. Biol. 9:690-695 (1999)]. Amino acid residues in thecorresponding loops, however, have been shown to interact withphospholipids in the PTB domain of Shc and in the structurallyhomologous PH domains of several signaling molecules [Lemmon et al.,Cell 85:621-624 (1996)]. Remarkably, SNT-1 is also capable ofinteracting with tyrosine-phosphorylated TRKs, which possess no sequencehomology to hFGFR1 (see below), illustrating the unique functionaldiversity of this conserved PTB domain fold.

Structural Insights into SNT Binding to Tyrosine-Phosphorylated TRKs:The new SNT-1 PTB domain structure yields insights into how the proteinmight interact with tyrosine-phosphorylated TRKs. Conserved Arg-63 andArg-78 residues in SNT-1 are structurally analogous to Arg-212 andArg-227 of IRS-1 PTB domain, respectively (FIGS. 6B and 6C). The latterpair of Arg residues are essential for IRS-1 binding to phosphotyrosinein the canonical NPXpY motif [Zhou et al., Nat. Struc, Biol. 3:388-393(1996)]. The mutagenesis and yeast two-hybrid binding studies disclosedherein show that the mutation of Arg-63 and Arg-78 to Gln results incomplete elimination of SNT-1 interaction with TRKB, but has no effecton binding to hFGFR1.

Additionally, Ala substitution of lie (pY-5) in the TRKA peptide(HIIENPQpYFSDA, which is highly homologous to TRKB) caused markedreduction in binding to the PTB domain. Based on these results, themechanism by which SNT-1 binds to TRKs is appears similar to that ofIRS-1 PTB domain interactions with NPXpY-containing proteins (FIG. 6C).Specifically, the phosphotyrosine of the NPXpY motif in TRKs wouldcoordinate with Arg-63 and Arg-78 in SNT-1 PTB domain, and residuesN-terminal to the phosphotyrosine would adopt an extended conformationwith hydrophobic side-chains intercalating into the hydrophobic pocketbetween β5 and α1, which is also a site for interactions with hFGFR1.Furthermore, NMR titration experiments showed that hFGFR1 and TRKpeptides compete for binding to the PTB domain, which agrees with theresults of the peptide competition experiments in a SNT-1 GST pull-downassay [Ong et al., Mol. Cell. Biol. 20:979-989 (2000)]. Together, theseresults demonstrate that the binding of SNT-1 to eithernon-phosphorylated FGFRs or tyrosine-phosphorylated TRKs is mutuallyexclusive.

Regulation of SNT and FGFR Association by a Possible LocalConformational Change: The β8 strand is structurally unique for the PTBdomain fold. It was therefore determined whether it was functionallyimportant for SNT-1 binding to hFGFR1 or phosphorylated TRKs. Truncationstudies of SNT-1 were therefore performed to address this question.Yeast two-hybrid assays showed that a truncated SNT-1 PTB domain lackingthe β8 region (residues 2-111) almost abolished its ability to interactwith hFGFR1, without decreasing its TRKB binding (FIG. 6C). AnotherSNT-1 truncation mutant (residues 11-114), which ends with β8 shortenedby one residue, showed markedly reduced binding to hFGFR1 peptide in NMRbinding studies (supported by significant line-broadening of the proteinNMR signals), but did not impair its interactions with thetyrosine-phosphorylated TRKB peptide. The effects of the β8 truncationwere further confirmed in ITC measurements of the SNT PTB domain bindingto hFGFR1 or TRK peptides. These results indicate that both the presenceand structural integrity of the β8 region are necessary for SNT-1binding to FGFRs but not for its interaction with TRKs. While theoverall structural fold of the SNT-1 PTB domain may be similar in itsfree, TRK- or hFGFR1-bound forms, the structural requirement of β8 isunique for its hFGFR1 association. This observation is consistent withthe fact that the anti-parallel β′ strand of the hFGFR1 peptide (7residues, FIG. 6A) is much longer than these NPXpY or related sequencesthat are recognized by the PTB domains of Shc [Zhou et al., J. Biol.Chem. 270:31119-31123 (1995)], IRS-1 [Zhou et al., Nature Struct. Biol.3:388-393 (1996), Eck et al., Cell 85:695-705 (1996)], or Numb [Zwahlenet al., EMBO J 19:1505-1515 (2000)]. These findings imply thatconformational perturbation of β8 would compromise SNT-1 binding toFGFRs, indicating that this β8 strand could act as an on/off switch forSNT-1/FGFR association.

Possible Role of SNTs as Molecular Switches in FGFR and TRK Signaling:The present structural and mutational analyses of the SNT-1 PTB domainsuggest mechanisms by which SNTs coordinate FGF and neurotrophinsignaling during neuronal differentiation. The ability of SNTs tointeract with non-phosphorylated FGFR raises the possibility that SNTsare sequestered by FGFRs in unstimulated cells and are only availablefor activation by FGFs. Events that trigger release of SNTs fromconstitutive FGFR association would allow for SNT interaction with TRKs,leading to a neurotrophin-responsive state in differentiating neurons.Such events may include FGF receptor down-regulation or conformationalperturbation to the β8 region of the SNT PTB domain bypost-translational modifications or interactions with other protein(s).Alternatively, even in the absence of SNT/FGFR complexes, theconformational flexibility of the SNT PTB domain may be constrainedduring neurogenesis to regulate its availability for interaction withand activation by neurotrophin receptors. These models may provide newclues to explain how differentiating neuronal precursors undergo thewell-documented switch from FGF dependence to neurotrophin dependence[Birren and Anderson Neuron 4:189-201 (1990); Ip et al., Neuron13:443-455 (1994); and Stemple et al., Neuron 1:517-525 (1988)], whichis not simply due to changes in TRK expression [Ip et al., Neuron13:443-455 (1994)].

Conclusion

The new three-dimensional structure of the SNT-1 PTB domain/hFGFR1complex reveals the unique features that enable SNT-1 to recognize tworadically different receptor sequences in a mutually exclusive manner.The results demonstrate that both adaptive hydrophobic interactions aswell as complementary electrostatic interactions are important factorsthat underlie specificity and versatility of molecular recognition bythe conserved PTB domain structural fold. These findings furtherindicate that cellular events, which cause a local conformational changein the PTB domain, may govern SNT interactions with either FGFRs orTRKs. Thus, the intrinsic adaptability and flexibility of the SNT PTBdomain appears to serve as a focal point for the essential interplaybetween FGF and neurotrophin receptor signaling that governs neuronalsurvival and differentiation.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which arehereby incorporated by reference herein in their entireties.

1. An isolated nucleic acid encoding a polypeptide comprising amino acidresidues 11-140 of SEQ ID NO:1, or amino acid residues 11-140 of SEQ IDNO:1 with a conservative amino acid substitution.
 2. The isolatednucleic acid of claim 1 further comprising a heterologous nucleotidesequence.
 3. An isolated nucleic acid encoding a peptide derived fromFGFR1 consisting of 16 to 50 amino acids comprising the amino acidsequence of SEQ ID NO:5: Val Xaa Xaa Leu Xaa Xaa Xaa Ile Xaa Leu Xaa ArgXaa Val Xaa Val; wherein said peptide binds to the PTB domain of SNT1.4. The isolated nucleic acid of claim 3 further comprising aheterologous nucleotide sequence.
 5. An isolated nucleic acid encoding apeptide derived from FGFR1 consisting of 16 to 50 amino acids comprisingthe amino acid sequence of SEQ ID NO:3 or SEQ ID NO:3 with aconservative amino acid substitution; wherein the peptide can bind tothe PTB domain of SNT1.
 6. The isolated nucleic acid of claim 5 furthercomprising a heterologous nucleotide sequence.
 7. A polypeptidecomprising the amino acid residues 11-140 of SEQ ID NO:1, or amino acidresidues 11-140 of SEQ ID NO:1 with a conservative amino acidsubstitution.
 8. A fusion protein or peptide comprising the polypeptideof claim
 7. 9. An isolated peptide derived from FGFR1 consisting of 16to 50 amino acids comprising the amino acid sequence of SEQ ID NO:5: ValXaa Xaa Leu Xaa Xaa Xaa Ile Xaa Leu Xaa Arg Xaa Val Xaa Val; wherein thepeptide can bind to the PTB domain of SNT1.
 10. A fusion protein orpeptide comprising the peptide of claim
 9. 11. An isolated peptidederived from FGFR1 consisting of 16 to 50 amino acids comprising theamino acid sequence of SEQ ID NO:3 or SEQ ID NO:3 with a conservativeamino acid substitution; wherein said peptide can bind to the PTB domainof SNT1.
 12. A fusion protein or peptide comprising the peptide of claim11.
 13. A method of identifying a compound that stabilizes a SNT/FGFRcomplex using the three-dimensional structure of the SNT/FGFR complexcomprising: (a) selecting a potential compound by performing rationaldrug design with the set of atomic coordinates obtained from Tables 1-5,wherein said selecting is performed in conjunction with computermodeling; (b) contacting the potential compound with a SNT/FGFR complexcomprising an SNT or an SNT fragment, and FGFR or an FGFR fragment; and(c) measuring the stability of the SNT/FGFR complex; wherein a potentialcompound is identified as a compound that stabilizes the SNT/FGFRcomplex when there is an increase in the stability of the SNT/FGFRcomplex.
 14. A method of identifying a compound that destabilizes aSNT/FGFR complex using the three-dimensional structure of the SNT/FGFRcomplex comprising: (a) selecting a potential compound by performingrational drug design with the set of atomic coordinates obtained fromTables 1-5, wherein said selecting is performed in conjunction withcomputer modeling; (b) contacting the potential compound with a SNT/FGFRcomplex comprising an SNT or an SNT fragment, and FGFR or an FGFRfragment; and (c) measuring the stability of the SNT/FGFR complex;wherein a potential compound is identified as a compound thatdestabilizes the SNT/FGFR complex when there is a decrease in thestability of the SNT/FGFR complex.
 15. A method of identifying acompound that inhibits the formation of a SNT/FGFR complex using thethree-dimensional structure of the SNT/FGFR complex comprising: (a)selecting a potential compound that binds to the PTB domain of SNT;wherein said selecting is performed using rational drug design with theset of atomic coordinates obtained from Tables 1-5, and is performed inconjunction with computer modeling; (b) contacting the potentialcompound with an SNT or an SNT fragment, and FGFR or an FGFR fragmentunder conditions in which the SNT/FGFR complex can form in the absenceof the potential compound; and (c) measuring the binding affinity of theSNT or the SNT fragment with FGFR or the FGFR fragment; wherein apotential compound is identified as a compound that inhibits theformation of the SNT/FGFR complex when there is a decrease in thebinding affinity of the SNT or the SNT fragment with FGFR or the FGFRfragment.
 16. A method of identifying a compound that stabilizes aSNT/FGFR complex comprising: (a) obtaining a set of atomic coordinatesdefining the three-dimensional structure of a SNT/FGFR complexconsisting of a fragment of SNT consisting of amino acid residues 11-140of SEQ ID NO:1 and a fragment of FGFR consisting of SEQ ID NO:3; (b)selecting a potential compound by performing rational drug design withthe atomic coordinates obtained in step (a), wherein said selecting isperformed in conjunction with computer modeling; (c) contacting thepotential compound with a SNT/FGFR complex; wherein said SNT/FGFRcomplex comprises an SNT or an SNT fragment, and FGFR or an FGFRfragment; and (d) measuring the stability of the SNT/FGFR complex ofstep (c); wherein a potential compound is identified as a compound thatstabilizes the SNT/FGFR complex when there is an increase in thestability of the SNT/FGFR complex of step (c).
 17. A method ofidentifying a compound that destabilizes a SNT/FGFR complex comprising:(a) obtaining a set of atomic coordinates defining the three-dimensionalstructure of a SNT/FGFR complex consisting of a fragment of SNTconsisting of amino acid residues 11-140 of SEQ ID NO:1 and a fragmentof FGFR consisting of SEQ ID NO:3; (b) selecting a potential compound byperforming rational drug design with the atomic coordinates obtained instep (a), wherein said selecting is performed in conjunction withcomputer modeling; (c) contacting the potential compound with a SNT/FGFRcomplex; wherein said SNT/FGFR complex comprises an SNT or an SNTfragment, and FGFR or an FGFR fragment; and (d) measuring the stabilityof the SNT/FGFR complex of step (c); wherein a potential compound isidentified as a compound that stabilizes the SNT/FGFR complex when thereis a decrease in the stability of the SNT/FGFR complex of step (c). 18.(canceled)
 19. A method of selecting a compound that potentiallyinhibits the SNT/FGFR dependent cellular signaling pathway comprising:(a) defining the structure of the SNT/FGFR complex by the atomiccoordinates obtained from Tables 1-5; and (b) selecting a compound whichpotentially inhibits the SNT/FGFR dependent cellular signaling pathway;wherein said selecting is performed with the aid of the structuredefined in step (a).
 20. A method of selecting a compound thatpotentially stimulates the SNT/FGFR dependent cellular signaling pathwaycomprising: (a) defining the structure of the SNT/FGFR complex by theatomic coordinates obtained from Tables 1-5; and (b) selecting acompound which potentially stimulates the SNT/FGFR dependent cellularsignaling pathway; wherein said selecting is performed with the aid ofthe structure defined in step (a).
 21. A method of selecting a compoundthat potentially binds to the PTB domain of SNT1 or the SNT/FGFR complexcomprising: (a) defining the structure of the SNT/FGFR complex by theatomic coordinates obtained from Tables 1-5; and (b) selecting acompound which potentially binds the PTB domain of SNT1 or the SNT/FGFRcomplex; wherein said selecting is performed with the aid of thestructure defined in step (a).
 22. A computer comprising arepresentation of a SNT/FGFR complex in computer memory which comprises:(a) a machine-readable data storage medium comprising a data storagematerial encoded with machine-readable data, wherein said data comprisesstructural coordinates from Tables 1-5; (b) a working memory for storinginstructions for processing said machine-readable data; (c) a centralprocessing unit coupled to said working memory and to saidmachine-readable data storage medium for processing said machinereadable data into a three-dimensional representation of the SNT/FGFRcomplex; and (d) a display coupled to said central-processing unit fordisplaying said three-dimensional representation.